Enhanced immune cell receptor sequencing methods

ABSTRACT

Disclosed are methods for sequencing immune cell receptor repertoires from immune cell populations, the methods comprising isolating RNA from immune cells, generating cDNA from the RNA, ligating adapter sequences to the cDNA, and sequencing the cDNA. Also provided are kits containing primer mixtures for the sequencing of immune cell receptor repertoires.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Patent Application Serial No. PCT/US2018/015819, filedJan. 30, 2018, which claims priority under 35 U.S.C. § 119(e) to U.S.provisional patent application Ser. No. 62/452,409, filed Jan. 31, 2017,the entire contents of each of which are incorporated herein byreference.

FIELD

The disclosure relates to methods, systems and kits for sequencingimmune cell receptor repertoires from immune cells, such as T-cells orB-cells.

BACKGROUND

Immune cell repertoires, such as B- or T-cell repertoires, consists ofmillions of lymphocytes, each expressing a different protein complexthat enables specific recognition of a single antigen. CD4 and CD8positive T-cells express so-called T-cell receptors (TCRs). Theseheterodimeric receptors recognize antigen-derived peptides displayed bymajor histocompatibility complex (MHC) molecules on the surface ofantigen presenting cells, as described in Rudolph M G, Stanfield R L,Wilson I A. How TCRs bind MHCs, peptides, and coreceptors. Annu RevImmunol. 2006; 24:419-66. TCRs are composed of two subunits, mostcommonly of one α and one β chain. A less common type of TCR containsone γ and one δ chain.

Alpha (α) chains consists of a (variable) V, a joining (J) and aconstant (C) region, while beta (β) chains contain an additionaldiversity (D) region between the V and the J region (see FIG. 1), asdescribed in Starr T K, Jameson S C, Hogquist K A. Positive and negativeselection of T cells. Annu Rev Immunol. 2003; 21:139-76. Each of theseTCR regions is encoded in several pieces, so-called gene segments, whichare spatially segregated in the germline. In humans, the TCR α genelocus contains 54 different V gene segments, and 61 J gene segments. Thehuman TCR β chain locus comprises 65 V, 2 D and 14 J segments. The greatstructural diversity of TCRs is achieved by somatic recombination ofthese TCR gene segments during lymphocyte development in the thymus.During this process, several gene segments of each region type arerandomly selected and joined to form a rearranged TCR locus. Additionaljunctional diversity is created by the addition or removal ofnucleotides at the sites of recombination, as described in Krangel M S.Mechanics of T cell receptor gene rearrangement. Curr Opin Immunol. 2009April; 21(2):133-9. The process of V(D)J joining plays a critical rolein shaping the third hypervariable loops (also called complementarydetermining regions, CDR3s) of the TCR α and β chains. These regionsbind antigens and are essential for providing the high specificity ofantigen recognition that TCRs exhibit.

Similarly to the TCR αβ, TCR gamma (γ) and delta (δ) segments undergoV(D)J rearrangement during thymus development. Both loci are recombinedin the double negative (DN) stage of T-cell development. Differentiationtowards γδ or αβ lineage relies on the ability of the cell to producefunctional γδ or αβ TCR. The δ locus is embedded within the α locus. Dδ,Jδ and Cδ segments are located in between the V and the J segment of theα locus. The Vδ segments are the same as the Vα segments but only afraction of the Vα segments are used for the TCR δ chain.

Overall, V(D)J recombination is able to generate millions of differentTCR sequences and plays a critical role in an organism's ability toeliminate infections or transformed cells. Not surprisingly, TCRrepertoires affect a wide range of diseases, including malignancy,autoimmune disorders and infectious diseases. TCR sequencing has beeninstrumental for our understanding of how the TCR repertoire evolvesduring infection or following treatment (e.g. after hematopoietic stemcell transplantation, chronical viral infection, immunotherapy).Further, the identification of TCRs on tumor-infiltrating lymphocytesand other T-cells that target cancer-specific epitopes has not onlyfurthered our knowledge of malignant disease, but has also led to noveltherapies for cancer such as adoptive T-cell transfer or cancervaccines.

Due to the large diversity of sequences, determining TCR repertoires hasbeen challenging in praxis. In the last couple of years, next generationsequencing (NGS) has opened up new opportunities to comprehensivelyassess the extreme diversity of TCR repertoires, as described in GenoletR, Stevenson B J, Farinelli L, Osterås M, Luescher I F. Highly diverseTCRα chain repertoire of pre-immune CD8⁺ T cells reveals new insights ingene recombination. EMBO J. 2012 Apr. 4; 31(7):1666-78; Robins H S,Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, RiddellS R, Warren E H, Carlson C S. Comprehensive assessment of T-cellreceptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5;114(19):4099-107; Linnemann C, Heemskerk B, Kvistborg P, Kluin R J,Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S,Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z,Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, SpitsH, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M,Schumacher T N. High-throughput identification of antigen-specific TCRsby TCR gene capture. Nat Med. 2013 November; 19(11):1534-41;Turchaninova M A, Britanova O V, Bolotin D A, Shugay M, Putintseva E V,Staroverov D B, Sharonov G, Shcherbo D, Zvyagin I V, Mamedov I Z,Linnemann C, Schumacher T N, Chudakov D M. Pairing of T-cell receptorchains via emulsion PCR. Eur J Immunol. 2013 September; 43(9):2507-15.

Since most current TCR sequencing techniques require enrichment of TCRgenes for sequencing, the majority of methods include an amplificationstep, in which the nucleic acids encoding the individual TCRs areamplified. Therefore, one of the challenges of the TCR sequencingrelates to the ability of the technology to maintain the proportion ofeach TCR during the amplification. Thus, the ways in which TCR librariesare prepared have a strong impact on the quality and the reliability ofthe obtained sequencing results and on the conclusions than can be drawnfrom the data. Several approaches have been used to amplify and sequenceTCR repertoires in the past, each method with its own set of issues.

One frequently employed method for TCR sequencing is based on amultiplex PCR step, in which all the primers for the V and the Jsegments are mixed together to amplify all the possible V(D)Jrearrangements/combinations, as described in Robins H S, Campregher P V,Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H,Carlson C S. Comprehensive assessment of T-cell receptor beta-chaindiversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107.The main drawback of this technology is that the amplification is notquantitative: Because the efficiency of each primer pair varies, someTCR sequences are preferentially represented in the library.

Another TCR sequencing method uses a process called “DNA gene capture”to isolate TCR encoding DNA fragments, as described in Linnemann C,Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K,Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P,Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B,Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H,Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N.High-throughput identification of antigen-specific TCRs by TCR genecapture. Nat Med. 2013 November; 19(11):1534-41. However, since thismethod uses DNA rather than RNA, this method will also isolate V and Jsegments that have not yet undergone somatic rearrangement. As aconsequence, many of the obtained sequencing data are uninformative forTCR gene identification as they do not contain the V(D)J region ofrearranged TCR gene locus. Furthermore, using DNA instead of RNA for theTCR gene analysis may overestimate the diversity of the TCR repertoireas only one of the two β chains is expressed by the T-cells while theother gene is silenced (allelic exclusion).

A third method of TCR amplification is based on the 5′-Race PCRtechnology (SMARTer® Human TCR a/b Profiling Kit, Takara-Clontech). Inthis method, a nucleic acid adapter is added to the 5′-end of the cDNAduring the reverse transcription step. As a result, TCR products can besubsequently amplified with a single primer pair, with one primerbinding to the adapter at the 5′-end of the cDNA and the second primerbinding to the constant region near the 3′-end of the cDNA. One of thedisadvantages of this technique is that the amplification step willgenerate PCR fragments ranging between 500 and 600 bp. As the length ofthe V segment exceeds 400 bp it is actually not possible to sequence theV(D)J junction starting from the 5′-end using ILLUMINA® sequencingtechnology, which can generate sequencing reads of up to 300 bp only.Sequencing of the V/J junction is thus usually performed from theconstant region, crossing the J segment, the CDR3 region and part of theV segment. However, sequencing errors increase with the length of thesequencing read, and are thus most frequently introduced in the Vsegments—the region most challenging to correctly assign due to the highhomology between different V segments. Consequently, sequencing startingfrom the constant region may lead to a reduction in the number of Vsegments that can be identified unambiguously. While this caveat can beavoided by paired-end sequencing, such modification of the protocol willsignificantly increase the duration and cost associated with thismethod.

SUMMARY

With each of the current methods exhibiting significant shortcomings,there is thus a considerable need for a TCR sequencing technology thatprovides TCR repertoire data with high sensitivity and reliability.

Disclosed herein are methods and kits for sequencing of T-cell receptorrepertoires and other immune cell repertoires, such as B-cellrepertoires, with high sensitivity and reliability. In one embodiment,the methods include the steps of (1) providing RNA from T-cells, (2)transcribing RNA into complimentary RNA (cRNA), (3) reverse transcribingthe cRNA into cDNA while introducing a common adapter to the 5′ end ofthe cDNA products, (4) amplifying the cDNA using a single primer pair,(5) further amplifying with PCR products with a single primer pair whichintroduces adapters for next generation sequencing, wherein the firstprimer binds to the common adapter region, and wherein the second primerbinds to the constant region of the TCR gene, and (6) sequencing the PCRproducts. In one embodiment, the methods include the steps of (1)providing RNA from T-cells, (2) reverse transcribing the RNA into cDNA,(3) generating second strand cDNA while introducing a common adapter tothe 5′ end of the cDNA products, (4) amplifying the cDNA using a singleprimer pair, (5) further amplifying with PCR products with a singleprimer pair which introduces adapters for next generation sequencing,wherein the first primer binds to the common adapter region, and whereinthe second primer binds to the constant region of the TCR gene, and (6)sequencing the PCR products. These embodiments are also called SEQTRmethod (Sequencing T-cell Receptors). Also provided are kits containingprimer mixtures for the sequencing of T-cell receptor repertoires.Similar methods and kits for sequencing of B-cell receptor repertoiresare provided.

According to one aspect, methods for sequencing immune cell receptorgenes are provided. The methods include (1) providing RNA from immunecells; 2)(a) optionally transcribing the RNA into complementary RNA(cRNA), followed by reverse transcribing the cRNA into complementary DNA(cDNA) using one or more primers that comprise a first adapter sequence,wherein each 5′ end of the cDNA produced by reverse transcriptioncontains the first adapter sequence; (2)(b) if step (2)(a) is notperformed, reverse transcribing the RNA into complementary DNA (cDNA),followed by transcribing the cDNA into second strand cDNA using one ormore primers that comprise a first adapter sequence, wherein each 5′ endof the cDNA produced by transcribing the cDNA into second strand cDNAcontains the first adapter sequence; (3) amplifying the cDNA to producea first amplification product using a first primer pair comprising afirst primer that hybridizes to the first adapter sequence and a secondprimer that hybridizes to a constant region of immune cell receptorgene; (4) amplifying the first amplification product to produce a secondamplification product using a second primer pair, in which (i) a firstprimer of the second primer pair binds to the adapter sequence at the 5′end of the second amplification product, (ii) the second primer of thesecond primer pair binds to the constant region of immune cell receptorgene in the second amplification product, and (iii) the first and secondprimers comprise adapter sequences for sequencing; and (5) sequencingthe second amplification product.

In some embodiments, the reverse transcription step results in PCRproducts ranging from 150-600 bp. In some embodiments, the immune cellreceptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR)genes.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) hybridize to TCR α chain V segments. In some embodiments, theone or more primers used for reverse transcription (step (2)(a)) orsecond strand cDNA synthesis (step (2)(b)) comprise one or more of SEQID NOs: 1-50 or SEQ ID NOs: 261-310.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) hybridize to TCR β chain V segments. In some embodiments, theone or more primers used for reverse transcription (step (2)(a)) orsecond strand cDNA synthesis (step (2)(b)) comprise one or more of SEQID NOs: 51-100 or SEQ ID NOs: 311-360.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) hybridize to TCR γ chain V segments.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) hybridize to TCR δ chain V segments.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) hybridize to BCR heavy chain V segments.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) hybridize to BCR light chain V segments.

In some embodiments, the one or more primers used for reversetranscription (step (2)(a)) or second strand cDNA synthesis (step(2)(b)) contain a nucleotide barcode sequence. In some embodiments, thenucleotide barcode comprises 6 to 20 nucleotides. In some embodiments,the nucleotide barcode consists of 9 nucleotides. In some embodiments,the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN orHHHHHNNNN.

In some embodiments, the first adapter sequence of the one or moreprimers used for the reverse transcription (step (2)(a)) or secondstrand cDNA synthesis (step (2)(b)) comprises a T7 adapter or anILLUMINA® adapter.

In some embodiments, the immune cells are T-cells and wherein the secondprimer of the first pair of primers hybridizes to the constant region ofa TCR gene.

In some embodiments, the immune cells are B-cells and wherein the secondprimer of the first pair of primers hybridizes to the constant region ofa BCR gene.

In some embodiments, the sequencing is next generation sequencing.

In some embodiments, the RNA from the immune cells is obtained by mixingimmune cells with carrier cells before RNA extraction.

In some embodiments, the immune cells are tumor-infiltratinglymphocytes.

In some embodiments, the immune cells are CD4 or CD8 positive T-cells.

In some embodiments, the immune cells are purified from peripheral bloodmononuclear cells (PBMC) before RNA extraction.

In some embodiments, the immune cells are part of a mixture of PBMC.

In some embodiments, the immune cells are derived from a mammal. In someembodiments, the mammal is a human or a mouse.

According to another aspect, kits for sequencing of T-cell receptors areprovided. The kits include at least one primer which comprises a TCR αchain V segment portion of any one of SEQ ID NOs: 1-50 or SEQ ID NOs:261-310 and a barcode sequence. In some embodiments, the kits include atleast one primer including any one of SEQ ID NOs: 1-50 or

SEQ ID NOs: 261-310.

According to another aspect, kits for sequencing of T-cell receptors areprovided. The kits include at least one primer which comprises a TCR τ3chain V segment portion of any one of SEQ ID NOs: 51-100 or SEQ ID NOs:311-360 and a barcode sequence. In some embodiments, the kits include atleast one primer comprising any one of SEQ ID NOs: 51-100 or SEQ ID NOs:311-360.

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the arrangement of (variable) V, diversity (D), joining (J)and constant (C) regions in the α and β chains of T-cell receptors.Figure taken from Murphy, K., Travers, P., Walport, M., & Janeway, C.(2012). Janeway's immunobiology. New York: Garland Science.

FIG. 2 is an illustration of three different TCR sequencing techniquesthat have been employed in the past.

FIG. 3 provides an overview of the SEQTR method, using TCR α chains asan example. Each bar represents a TCR α chain gene. In RNA and cRNAmolecules, the order of the segments is, left to right: V segments, Jsegments, and the constant region. Barcode regions are added in cDNAmolecule to the left of V segments; and T7 adapter regions are added tothe left of the barcodes (also indicated by T7 primer amplification inPCR1 and PCR2 steps). ILLUMINA® sequencing adapters are added in thePCR2 step to the 5′ and 3′ ends of the molecules, as shown in the lastset of molecules.

FIG. 4 illustrates the sensitivity of the SEQTR method. 10{circumflexover ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4,10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, weremixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extractedand subjected to transcription, reverse transcription and one round ofamplification (steps 2-4, see Detailed Description). The resulting PCRproducts were separated on an agarose gels and visualized with ethidiumbromide.

FIG. 5 illustrates the specificity of the SEQTR method. 10{circumflexover ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4,10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, weremixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extractedand subjected to the SEQTR method. The percentages of sequencing readsthat were or were not, respectively, associated with actual TCR genesare indicated.

FIG. 6 illustrates the unambiguous identification of TCR genes as afeature of the SEQTR method. 5×10{circumflex over ( )}4 3T3 cells weremixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5,10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positiveT-cells, respectively. The RNA of each mixture was isolated andsubjected to the SEQTR method. Reads that were not associated with TCRgenes were removed from the data set. For the remaining reads, thepercentages of reads that could or could not, respectively, beunambiguously assigned to specific V or J segments are indicated.

FIG. 7 illustrates the linearity of the SEQTR method. A fixed amount ofDNA encoding a known TCR sequence was diluted at differentconcentrations into a DNA mixture representing a naïve CD8 T-cellrepertoire. TCR repertoires of the individual mixtures were sequencedusing the SEQTR method. The observed frequency of the known TCR sequencein the entire repertoire was plotted against the respective TCR genedilution.

FIG. 8 illustrates the reproducibility of the SEQTR method. TCRrepertoires were sequenced using the SEQTR method from one biologicalsample in two independent technical replicates. The frequencies for eachV-J rearrangement/combination in TCR β chains were determined andcompared between the two replicates. Each sphere represents a single V-Jrearrangement with the size of a sphere indicating the relativefrequency of the specific V-J recombination. Grey spheres representrearrangements for which the relative frequencies detected in the tworeplicates differed by less than two-fold. Black spheres representrearrangements for which the relative frequencies detected in the tworeplicates differed by more than two-fold.

FIGS. 9A-9C illustrates the diversity of three different TCR repertoiresequencing data set using the SEQTR method. FIG. 9A CD8 positive T-cellswere isolated from peripheral blood mononuclear cells (PBMC), FIG. 9Badditionally purified using tetramers conjugated with neo-epitopeTEDYMIHII (SEQ ID NO:236) or FIG. 9C additionally purified usingtetramer conjugated with neo-epitope (same as in FIG. 9B) andsubsequently expanded in vitro. The TCR repertoires of the respectivesamples were sequenced using the SEQTR method and the relativefrequencies of all observed V/J rearrangements/combinations plotted.

FIGS. 10A-10C illustrate the overlap of TCRs identified using a singlecell cloning method and TCRs identified using the SEQTR method. FIG.10A: T-cells were isolated from PBMC and subjected to an additionalround of purification using tetramers conjugated with neo-epitopeTEDYMIHII (SEQ ID NO: 236). The resulting cell population was thensorted by fluorescence-activated cell sorting (FACS). Half of the sortedcells were subjected to the SEQTR method to sequence the TCR repertoire.For the other half of cells, individual T-cell clones were isolated andexpanded in vitro (single cell cloning). Once the clones wereestablished, the TCR genes of each T-cell clones were amplified andsequenced using classical Sanger sequencing. FIG. 10B: The table showsall six TCRs identified using the single cell cloning method. Thesequences correspond to SEQ ID NOs: 237 through 242 from top to bottom,respectively. FIG. 10C: The table shows the eight most frequent TCRsidentified using the SEQTR method. The sequences correspond to SEQ IDNOs: 243 through 250 from top to bottom, respectively.

FIG. 11 illustrates the number of reads for different samples obtainedusing the TCR sequencing service “immunoSEQ®” offered by AdaptiveBiotechnology. The requested number of reads per sample was 200,000reads. The number on the x axis represent analysis of samples from 16different patients. Columns, left to right, for each sample representnumber of reads from: the tumor; the stroma (tissue surrounding thetumor); epitope specific TIL (Tumor Infiltrating Lymphocyte) stainedwith tetramer and sorted by FACS from the tumor sample (TET); andtetramer sorted TIL from a piece of the tumor that has been engrafted ina mice (mTET).

FIG. 12 illustrates the amplification of TCR genes from T-cells that arepart of a PBMC mixture (upper panel) or from isolated, CD4 positiveT-cells (lower panel), using steps 2 to 4 of the SEQTR method.

DETAILED DESCRIPTION

In light of the shortcomings of existing techniques to sequence TCRs, itwas determined that a TCR sequencing technology providing the mostreliable TCR repertoire data includes the following features:

-   -   1) The amplification of TCR genes is linear and does not employ        multiplex PCR, therefore avoiding artificial overrepresentation        of certain TCR sequences.    -   2) The method is based on RNA and not DNA, thus only providing        data for TCR sequences that have undergone rearrangement and        that are actually expressed in T-cells.    -   3) TCR genes are sequenced from the 5′ end, providing high        quality sequencing data and therefore maximizing reliable and        unambiguous identification of the highly homologous V segments.    -   4) Sequencing data include the highly variable CDR3 region,        therefore facilitating unambiguous identification of TCR        sequences.

The disclosed methods, systems and kits fulfill all these criteria.These same features are of use in sequencing receptors from other immunecells, such as B-cells.

In some embodiments, the immune cell receptor sequencing methodscomprise the following steps:

-   -   (1) Providing total RNA (RNA) as the starting material;    -   (2)(a) Transcribing the RNA into complimentary RNA (cRNA)        followed by reverse transcribing the cRNA into cDNA, using        primers that introduce a common adapter to the 5′ end of the        cDNA products;    -   (2)(b) If step (2)(a) is not performed, reverse transcribing the        RNA into complementary DNA (cDNA), followed by transcribing the        cDNA into second strand cDNA using one or more primers that        comprise a first adapter sequence, wherein each 5′ end of the        cDNA produced by transcribing the cDNA into second strand cDNA        contains the first adapter sequence;    -   (3) Amplifying the cDNA products using a single primer pair;    -   (4) Amplifying the PCR products of step 4 using a single primer        pair, in which:        -   i. the primers introduce adapters for next generation            sequencing, and        -   ii. the first primer binds to the common adapter region at            the 5′ end of the PCR products, and        -   iii. the second primer binds to a region of the PCR products            that constitutes the constant region of the TCR to be            sequenced; and    -   (5) Sequencing the PCR products generated in step 4.        Genetic Information to be Sequenced

The genetic information to be sequenced is immune cell receptor genes.In the some embodiments of the invention, the genetic information to besequenced comprises T-cell receptors genes. In some embodiments, the TCRgenes that are sequenced encode TCR α chains or TCR β chains. In otherembodiments, TCR genes that are sequenced encode TCR δ chains or TCR γchains.

In other embodiments of the invention, the genetic information to besequenced comprises B-cell receptor (BCR) genes.

Starting Material (Step 1)

RNA is isolated from immune cells and used to generate complimentary RNA(cRNA) by in vitro transcription. This is in contrast to existing TCRsequencing techniques that use DNA or complementary DNA (cDNA) as theirgenetic starting material.

In some embodiments, the immune cells from which RNA is obtained areisolated from peripheral blood mononuclear cells before RNA extraction.The immune cells are, in some embodiments, T-cells or B-cells.

In some embodiments, T-cells from which RNA is obtained express CD4 orCD8.

Generation of cRNA Through Transcription (Step (2)(a))

Complementary RNA (cRNA) is generated by in vitro transcription. Anymethod for performing in vitro transcription known to those skilled inmolecular biology can be used. In some embodiments, the in vitrotranscription in step 2 is performed using commercially available kits,such as the AMBION™ kits available from Thermo Fisher Scientific.

Reverse Transcription (Step (2)(a))

Reverse transcription of the cRNA is performed to generate complementaryDNA (cDNA). Methods known to persons skilled in molecular biology areused to reverse transcribe cRNA to cDNA. Typically, such methods includehybridization of a primer to the 3′ end of the cRNA molecule andproduction of DNA starting at the hybridized primer using a reversetranscriptase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the reverse transcription reaction isimportant for the ability to differentiate between homologous, yetdistinct, immune cell receptor sequences with high degrees of certaintyand allows shortening of the V segments from the 5′ end, generating PCRproducts with a size of 250-300 bp. Such a size range of PCR products isoptimal for next generation sequencing.

In some embodiments, the primers used for the reverse transcription aredesigned to bind within the V segments of the TCR genes (see FIG. 3).For example, the reverse transcription primers are designed to bindclose enough to the V(D)J junction so that the resulting sequencing datacover the CDR3 of the V segment and the J segment, but far enough fromthe V(D)J junction to still allow differentiation between different Vregions.

In some embodiments of the invention, a set of preferred primers is used(see, e.g., the sequences in Table 2 and Table 4, and Table 8 and Table9). Due to the high degree of homology between different V segments,some of the primers described in Table 2 and Table 4 (and Table 8 andTable 9) bind to more than one V segment (see Table 3 and Table 5; thebinding sites in their respective V segments for primers SEQ ID NOs:1-100 and SEQ ID NOs: 261-360 are indicated in Table 15 and Table 16).However, the design of the primers presented in Table 2 and Table 4(likewise Table 8 and Table 9) still allows the unambiguousassignment/identification of the respective V segments based ondifferences between the V segments downstream of the primer-bindingsite. In an alternative embodiment of the invention, only a subset ofthe preferred primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 may beused for the reverse transcription.

In yet another embodiment of the invention, primer sets may be used thatbind to different regions in the V segments when compared to the primershaving SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. For instance, thebinding site of one or more primers may be moved towards the CDR3 regionof the TCR gene. Due to the high degree of homology between V segments,the further the primer binding site is moved in the direction of theCDR3 region of the TCR gene, the larger the likelihood that theresulting sequencing data are consistent with the presence of more thanone V segment. While, in these cases, the respective V segments cannotbe assigned or identified unambiguously, the number of V/J segmentspossibly present in the sample can often be narrowed down to a smallsubset. Depending on the application, such limited information canalready be of value to the experimenter.

In another embodiment of the invention, the binding site of one or moreprimers may be moved towards the 5′ end of the V segment as compared tothe binding sites of primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360.Many next generation sequencing technologies generate sequencing readsthat are 150 bp long. Therefore, the further the primer binding site ismoved towards the 5′ end of the V segment, the larger is the probabilitythat the respective J segment (which can be found at the 3′ end of theresulting sequencing read) cannot be identified unambiguously. However,this problem can be circumvented by using alternative sequencingtechnologies that generate reads >150 bp.

In some embodiments, the primers used in step (2)(a) additionallycontain a unique bar code. Such barcoding of each RNA molecule beforethe amplification can be used to correct the obtained sequencing resultsfor PCR and sequencing errors.

In some embodiments, the primers for this reverse transcription stepintroduce a common T7 adapter at the 5′ end of the resulting PCRproducts. However, alternative adapter sequences are possible,including, but not limited to ILLUMINA® adapters and sequences presentedin Table 1.

TABLE 1 Examples for alternative nucleotide adapters that can be usedinstead of a T7 adapter sequence SEQ ID NO Primer namePrimer sequence (5′ to 3′) 251 Original EberwineAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCT T7 252 Affymetrix T7GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGT 253 Invitrogen T7TAATACGACTCACTATAGGGAGGCGGT 254 Ambion T7 GGTAATACGACTCACTATAGGGAGAAGAGT255 Agilent T7 AATTAATACGACTCACTATAGGGAGATReverse Transcription (Step (2)(b))

Reverse transcription of the RNA is performed to generate complementaryDNA (cDNA). Methods known to persons skilled in molecular biology areused to reverse transcribe RNA to cDNA. Typically, such methods includehybridization of a primer to the 3′ end of the RNA molecule andproduction of DNA starting at the hybridized primer using a reversetranscriptase enzyme and appropriate nucleotides, salts and buffers.

Transcribing the cDNA into Second Strand cDNA (Step (2)(b))

Following generation of cDNA, second strand cDNA is synthesized usingmethods known to persons skilled in molecular biology. Typically, suchmethods include hybridization of a primer to the 3′ end of the cDNAmolecule and production of second strand cDNA starting at the hybridizedprimer using a polymerase enzyme and appropriate nucleotides, salts andbuffers.

The choice of primers used in the second strand synthesis reaction isstep (2)(b) is as described above for reverse transcription in step(2)(a). The choice of primers is important for the ability todifferentiate between homologous, yet distinct, immune cell receptorsequences with high degrees of certainty and allows shortening of the Vsegments from the 5′ end, generating PCR products with a size of 250-300bp. Such a size range of PCR products is optimal for next generationsequencing.

Amplification (Step 3)

Amplification of the cDNA is performed by any of the well-knownamplification reactions, such as polymerase chain reaction (PCR).Methods known to persons skilled in the molecular biology art are usedto amplify the cDNA or a portion thereof (e.g., as depicted in FIG. 3).Typically, such methods include hybridization of a pair of primers tothe cDNA molecule and amplification of the DNA sequence between thehybridized primers using a polymerase enzyme and appropriatenucleotides, salts and buffers.

In some embodiments, the first primer of a primer pair used in anamplification step binds to the common adapter region of the cDNAproducts produced in step 3 and the second primer of the primer pairbinds to a region of the cDNA products that constitutes the constantregion of the TCR to be sequenced (see FIG. 3).

Of note, not all reverse primers designed to target the constant regionof the TCR gene perform equally well in this reaction. For example, theprimers listed in Table 7 all failed to provide good amplification withthe selected T7 5′ adapter. Therefore, in certain embodiments, theprimers listed in are Table 6 used in this amplification step.

Amplification (Step 4)

A second amplification step is performed to add additional sequences tothe amplified molecules, such as sequences that are useful in downstreamDNA sequencing reactions. In some embodiments of the present invention,the primers used in this step add appropriate adapters for ILLUMINA®sequencing.

Sequencing (Step 5)

Various suitable sequencing methods described herein or known in the artare used to obtain sequence information from the amplified sequencesfrom the nucleic acid molecules within a sample. For example, sequencingmethodologies that can be used in the methods disclosed herein include:classic Sanger sequencing, massively parallel sequencing, nextgeneration sequencing, polony sequencing, 454 pyrosequencing, ILLUMINA®sequencing, SOLEXA® sequencing, SOLID™ sequencing (sequencing byoligonucleotide ligation and detection), ion semiconductor sequencing,DNA nanoball sequencing, heliscope single molecule sequencing, singlemolecule real time sequencing, nanopore DNA sequencing, tunnelingcurrents DNA sequencing, sequencing by hybridization, sequencing withmass spectrometry, microfluidic Sanger sequencing, microscopy-basedsequencing, RNA polymerase sequencing, in vitro virus high-throughputsequencing, Maxam-Gilbert sequencing, single-end sequencing, paired-endsequencing, deep sequencing, and/or ultra-deep sequencing.

Definitions

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, unless the context clearly dictates otherwise, between theupper and lower limits of that range is also specifically disclosed.Each smaller range between any stated value or intervening value in astated range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange, and each range where either, neither, or both limits are includedin the smaller ranges is also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

As used herein, a “primer” is a nucleic acid molecule that hybridizeswith a complementary (including partially complementary) polynucleotidestrand. Primers can be DNA molecules, RNA molecules, or DNA or RNAanalogs. DNA or RNA analogs can be synthesized from nucleotide analogs.

EXAMPLES Example 1: Exemplary Protocol for the SEQTR Method Using T7 andTrueSeq Adapters

TCR α and β chain genes were sequenced in two independent reactions.

-   -   1) Starting material and RNA extraction        -   To obtain sufficient amounts of RNA in the extraction, a            minimum of 500,000 T-cells were used as starting material.            Alternatively, and especially in instances where fewer            T-cells were available, T-cells were mixed with 50,000 mouse            3T3 cells that served as carrier. T-cell RNA was extracted            using the RNeasy® Micro Kit from Qiagen Inc. according the            manufacturer's instruction with the following modification:            Elution was performed with 20 μl of water preheated to            50° C. RNA quality and quantity was verified using a            fragment analyzer.    -   2) cRNA synthesis by in vitro transcription (IVT):        -   In vitro transcription of isolated RNA was performed using            the MessageAmp™ II aRNA Amplification Kit from Ambion®            (Thermo Fisher Scientific), which contains enzymes, buffers            and nucleotides required to perform the first and second            strand cDNA and the in vitro transcription. The kit also            provides all columns and reagents needed for the cDNA and            cRNA purifications. RNA amplification was performed            according to the manufacturer's instructions with the            following modifications: 1) Between 0.5 and 1 μg of total            RNA as was used as starting material. 2) The IVT was            performed in a final volume of 40 μl, and incubated at            37° C. for 16 h. Purified cRNA was quantified by absorbance            using a NanoDrop™ spectrophotometer (Thermo Fisher            Scientific).    -   3) cDNA synthesis by reverse transcription:        -   The reverse transcription of the cRNA was performed with the            SuperScript® III from Invitrogen (Thermo Fisher Scientific).            The kit provides the enzyme, the buffer and the            dithiothreitol (DTT) needed for the reaction.            Deoxynucleotides (dNTPs) and RNAsin® Ribonuclease inhibitor            were purchased from Promega. The sequences for the primers            used for the reverse transcription can be found in Table 2            (primers for sequencing TCR α chain genes) and Table 4            (primers for sequencing TCR β chain genes).        -   500 ng of cRNA were used as starting material for the            reverse transcription. cRNA was mixed with 1 μl hTRAV or            hTRBV primers mix (2 μM each) and 1 μl dNTP (25 mM) in a            final volume of 13 μl. The mix was first incubated at 70° C.            for 10 min, then at 50° C. for 30 s. 4 μl 5× buffer, 1 μl            DTT (100 mM), 1 μl SuperScript III and 1 μl RNAsin® were            added to the mix. The samples were subsequently incubated            for at 55° C. 1 h and then at 85° C. for 5 min. After the            cDNA synthesis, 1 μg DNase-free RNase (Roche) was added to            the cDNA and incubated at 37° C. for 30 min to remove the            cRNA.    -   4) TCR gene amplification:        -   TCR gene amplification was performed using a Phusion®            High-Fidelity DNA polymerase (New England Biolabs) under the            following conditions:        -   PCR mix: 1 μl cDNA from step 3, 1 μl dNTPs (25 mM), 1 μl            primer mix (10 μM each, see Table 5), 5 μl 5× buffer and 0.2            μl Phusion® enzyme in a total volume of 25 μl.        -   PCR conditions:            -   94° C. for 5 min            -   20 to 30 cycles of                -   98° C. for 10 s                -   55° C. for 30 s                -   72° C. for 30 s            -   72° C. for 2 min        -   PCR products were purified either from agarose gels (using a            Qiaquick Gel Extraction Kit from Qiagen) or using an            ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) according to            the manufacturer's instructions.    -   5) Addition of Next Generation Sequencing adapters:        -   ILLUMINA® sequencing adapters were added by PCR using a            Phusion® High-Fidelity DNA polymerase (New England Biolabs).            One third of the purified PCR product obtained in step 4 was            mixed with 0.5 μl dNTPs (25 mM), 1 μl primer mix (10 μM            each, see Table 8), 5 μl 5× buffer and 0.2 μl Phusion®            enzyme in a total volume of 25 μl.        -   PCR conditions:            -   94° C. for 5 min            -   perform 12 cycles of:                -   98° C. for 10 s                -   55° C. for 30 s                -   72° C. for 30 s            -   72° C. for 2 min    -   6) TCR library purification:        -   10 μl of the PCR product from step 5 were purified using an            ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) or Ampure XP            beads (Beckman Coulter) according to the manufacturer's            instruction. Samples could then directly be used for            ILLUMINA® sequencing.

TABLE 2 Preferred primer sequences for amplification of TCR αchain V segments. N can beany nucleotide. The sequences for primers presented in this table consist ofthree parts (listed from 5′ to 3′): T7 adapter, barcode and TCR αchain V segment. SEQ Sequence Sequence ID Primer T7 adapter portion barcode portion Sequence TCR α chain V segment NO name of the primerof the primer portion of the primer  1 hTRAV1-1 TGTAATACGACTCACTATAGNNNNTNNNN CTTCTACAGGAGCTCCAGATGAAAG  2 hTRAV1-2 TGTAATACGACTCACTATAGNNNNTNNNN CTTTTGAAGGAGCTCCAGATGAAAG  3 hTRAV2 TGTAATACGACTCACTATAGNNNNTNNNN TGCTCATCCTCCAGGTGCGGGA  4 hTRAV3 TGTAATACGACTCACTATAGNNNNTNNNN GAAGAAACCATCTGCCCTTGTGA  5 hTRAV4 TGTAATACGACTCACTATAGNNNNTNNNN CCTGCCCCGGGTTTCCCTGAGCGAC  6 hTRAV5 TGTAATACGACTCACTATAGNNNNTNNNN TCTCTGCGCATTGCAGACACCCA  7 hTRAV6 TGTAATACGACTCACTATAGNNNNTNNNN TTGTTTCATATCACAGCCTCCCA  8 hTRAV7 TGTAATACGACTCACTATAGNNNNTNNNN GCTTGTACATTACAGCCGTGCA  9 hTRAV8-1/8-3 TGTAATACGACTCACTATAGNNNNTNNNN ATCTGAGGAAACCCTCTGTGCA 10 hTRAV8-2/8-4 TGTAATACGACTCACTATAGNNNNTNNNN ACCTGACGAAACCCTCAGCCCAT 11 hTRAV8-5 TGTAATACGACTCACTATAGNNNNTNNNN CCTATGCCTGTCTTTACTTTAATC 12 hTRAV8-6 TGTAATACGACTCACTATAGNNNNTNNNN CTTGAGGAAACCCTCAGTCCATAT 13 hTRAV8-7 TGTAATACGACTCACTATAGNNNNTNNNN GAAACCATCAACCCATGTGAGTGA 14 hTRAV9-1 TGTAATACGACTCACTATAGNNNNTNNNN ACTTGGAGAAAGACTCAGTTCAA 15 hTRAV9-2 TGTAATACGACTCACTATAGNNNNTNNNN ACTTGGAGAAAGGCTCAGTTCAA 16 hTRAV10 TGTAATACGACTCACTATAGNNNNTNNNN CTGCACATCACAGCCTCCCA 17 hTRAV11 TGTAATACGACTCACTATAG NNNNTNNNNGTTTGGAATATCGCAGCCTCTCAT 18 hTRAV12-1 TGTAATACGACTCACTATAG NNNNTNNNNCCCTGCTCATCAGAGACTCCAAG 19 hTRAV12-2 TGTAATACGACTCACTATAG NNNNTNNNNCTCTGCTCATCAGAGACTCCCAG 20 hTRAV12-3 TGTAATACGACTCACTATAG NNNNTNNNNCCTTGTTCATCAGAGACTCACAG 21 hTRAV13-1 TGTAATACGACTCACTATAG NNNNTNNNNTCCCTGCACATCACAGAGACCCAA 22 hTRAV13-2 TGTAATACGACTCACTATAG NNNNTNNNNTCTCTGCAAATTGCAGCTACTCAA 23 hTRAV14 TGTAATACGACTCACTATAG NNNNTNNNNTTGTCATCTCCGCTTCACAACTGG 24 hTRAV15 TGTAATACGACTCACTATAG NNNNTNNNNGTTTTGAATATGCTGGTCTCTCAT 25 hTRAV16 TGTAATACGACTCACTATAG NNNNTNNNNCCTGAAGAAACCATTTGCTCAAGA 26 hTRAV17 TGTAATACGACTCACTATAG NNNNTNNNNTCCTTGTTGATCACGGCTTCCCGG 27 hTRAV18 TGTAATACGACTCACTATAG NNNNTNNNNACCTGGAGAAGCCCTCGGTGCA 28 hTRAV19 TGTAATACGACTCACTATAG NNNNTNNNNCACCATCACAGCCTCACAAGTCGT 29 hTRAV20 TGTAATACGACTCACTATAG NNNNTNNNNTTTCTGCACATCACAGCCCCTA 30 hTRAV21 TGTAATACGACTCACTATAG NNNNTNNNNCTTTATACATTGCAGCTTCTCAGCC 31 hTRAV22 TGTAATACGACTCACTATAG NNNNTNNNNGTACATTTCCTCTTCCCAGACCAC 32 hTRAV23 TGTAATACGACTCACTATAG NNNNTNNNNCATTGCATATCATGGATTCCCAGC 33 hTRAV24 TGTAATACGACTCACTATAG NNNNTNNNNGCTATTTGTACATCAAAGGATCCC 34 hTRAV25 TGTAATACGACTCACTATAG NNNNTNNNNCAGCTCCCTGCACATCACAGCCA 35 hTRAV26-1 TGTAATACGACTCACTATAG NNNNTNNNNTTGATCCTGCCCCACGCTACGCTGA 36 hTRAV26-2 TGTAATACGACTCACTATAG NNNNTNNNNTTGATCCTGCACCGTGCTACCTTGA 37 hTRAV27 TGTAATACGACTCACTATAG NNNNTNNNNGTTCTCTCCACATCACTGCAGCC 38 hTRAV28 TGTAATACGACTCACTATAG NNNNTNNNNGCCACCTATACATCAGATTCCCA 39 hTRAV29 TGTAATACGACTCACTATAG NNNNTNNNNTCTCTGCACATTGTGCCCTCCCA 40 hTRAV30 TGTAATACGACTCACTATAG NNNNTNNNNCCCTGTACCTTACGGCCTCCCAGCT 41 hTRAV31 TGTAATACGACTCACTATAG NNNNTNNNNCTTATCATATCATCATCACAGCCA 42 hTRAV32 TGTAATACGACTCACTATAG NNNNTNNNNTCCCTGCATATTACAGCCACCCAA 43 hTRAV33 TGTAATACGACTCACTATAG NNNNTNNNNACCTCACCATCAATTCCTTAAAAC 44 hTRAV34 TGTAATACGACTCACTATAG NNNNTNNNNTCCCTGCATATCACAGCCTCCCAG 45 hTRAV35 TGTAATACGACTCACTATAG NNNNTNNNNCTTCCTGAATATCTCAGCATCCAT 46 hTRAV36 TGTAATACGACTCACTATAG NNNNTNNNNTCCTGAACATCACAGCCACCCAG 47 hTRAV37 TGTAATACGACTCACTATAG NNNNTNNNNTCCCTGCACATACAGGATTCCCAG 48 hTRAV38 TGTAATACGACTCACTATAG NNNNTNNNNCAAGATCTCAGACTCACAGCTGG 49 hTRAV39 TGTAATACGACTCACTATAG NNNNTNNNNCCGTCTCAGCACCCTCCACATCA 50 hTRAV40 TGTAATACGACTCACTATAG NNNNTNNNNCCATTGTGAAATATTCAGTCCAGG

TABLE 3 V segments targeted by each primer used for the amplification ofTCR α chain V segments. SEQ ID NO Primer Targeted V segment(s) 1hTRAV1-1 hTRAV01-1 2 hTRAV1-2 hTRAV01-2 3 hTRAV2 hTRAV02 4 hTRAV3hTRAV03 5 hTRAV4 hTRAV04 6 hTRAV5 hTRAV05 7 hTRAV6 hTRAV06 8 hTRAV7hTRAV07 9 hTRAV8-1/8-3 hTRAV08-1, hTRAV08-3 10 hTRAV8-2/8-4 hTRAV08-2,hTRAV08-4 11 hTRAV8-5 hTRAV08-5 12 hTRAV8-6 hTRAV08-6 13 hTRAV8-7hTRAV08-7 14 hTRAV9-1 hTRAV09-1 15 hTRAV9-2 hTRAV09-2 16 hTRAV10hTRAV10, hTRAV41 17 hTRAV11 hTRAV11 18 hTRAV12-1 hTRAV12-1 19 hTRAV12-2hTRAV12-2 20 hTRAV12-3 hTRAV12-3 21 hTRAV13-1 hTRAV13-1 22 hTRAV13-2hTRAV13-2 23 hTRAV14 hTRAV14 24 hTRAV15 hTRAV15 25 hTRAV16 hTRAV16 26hTRAV17 hTRAV17 27 hTRAV18 hTRAV18 28 hTRAV19 hTRAV19 29 hTRAV20 hTRAV2030 hTRAV21 hTRAV21 31 hTRAV22 hTRAV22 32 hTRAV23 hTRAV23 33 hTRAV24hTRAV24 34 hTRAV25 hTRAV25 35 hTRAV26-1 hTRAV26-1 36 hTRAV26-2 hTRAV26-237 hTRAV27 hTRAV27 38 hTRAV28 hTRAV28 39 hTRAV29 hTRAV29 40 hTRAV30hTRAV30 41 hTRAV31 hTRAV31 42 hTRAV32 hTRAV32 43 hTRAV33 hTRAV33 44hTRAV34 hTRAV34 45 hTRAV35 hTRAV35 46 hTRAV36 hTRAV36 47 hTRAV37 hTRAV3748 hTRAV38 hTRAV38-1, hTRAV38-2 49 hTRAV39 hTRAV39 50 hTRAV40 hTRAV40

TABLE 4 Preferred primer sequences for amplification of TCR βchain V segments. N can beany nucleotide. The sequences for primers presented in this table consist of threeparts (listed from 5′ to 3′): T7 adapter, barcode and TCR βchain V segment. SEQ Sequence Sequence ID Primer T7 adapter portion barcode portion Sequence TCR β chain V segment NO name of the primerof the primer portion of the primer  51 hTRBV1 TGTAATACGACTCACTATAGNNNNANNNN GTGGTCGCACTGCAGCAAGAAGA  52 hTRBV2 TGTAATACGACTCACTATAGNNNNANNNN GATCCGGTCCACAAAGCTGGAGGA  53 hTRBV3-1 TGTAATACGACTCACTATAGNNNNANNNN CATCAATTCCCTGGAGCTTGGTGA  54 hTRBV4-1 TGTAATACGACTCACTATAGNNNNANNNN TTCACCTACACGCCCTGCAGCCAG  55 hTRBV4-2 TGTAATACGACTCACTATAGNNNNANNNN TTCACCTACACACCCTGCAGCCAG  56 hTRBV5-1 TGTAATACGACTCACTATAGNNNNANNNN GAATGTGAGCACCTTGGAGCTGG  57 hTRBV5-2 TGTAATACGACTCACTATAGNNNNANNNN TACTGAGTCAAACACGGAGCTAGG  58 hTRBV5-3 TGTAATACGACTCACTATAGNNNNANNNN GCTCTGAGATGAATGTGAGTGCCT  59 hTRBV5-4 TGTAATACGACTCACTATAGNNNNANNNN CTGAGCTGAATGTGAACGCCTT  60 hTRBV6-1 TGTAATACGACTCACTATAGNNNNANNNN GAGTTCTCGCTCAGGCTGGAGT  61 hTRBV6-2 TGTAATACGACTCACTATAGNNNNANNNN CTGGGGTTGGAGTCGGCTGCTC  62 hTRBV6-4 TGTAATACGACTCACTATAGNNNNANNNN CCCCTCACGTTGGCGTCTGCTG  63 hTRBV6-5 TGTAATACGACTCACTATAGNNNNANNNN TCCCGCTCAGGCTGCTGTCGGC  64 hTRBV6-6 TGTAATACGACTCACTATAGNNNNANNNN GATTTCCCGCTCAGGCTGGAGT  65 hTRBV6-7 TGTAATACGACTCACTATAGNNNNANNNN TCCCCCTCAAGCTGGAGTCAGCT  66 hTRBV6-8 TGTAATACGACTCACTATAGNNNNANNNN TCCCACTCAGGCTGGTGTCGGC  67 hTRBV7-1 TGTAATACGACTCACTATAGNNNNANNNN CTCTGAAGTTCCAGCGCACACA  68 hTRBV7-2 TGTAATACGACTCACTATAGNNNNANNNN GATCCAGCGCACACAGCAGGAG  69 hTRBV7-3 TGTAATACGACTCACTATAGNNNNANNNN ACTCTGAAGATCCAGCGCACAGA  70 hTRBV7-5 TGTAATACGACTCACTATAGNNNNANNNN AGATCCAGCGCACAGAGCAAGG  71 hTRBV7-6 TGTAATACGACTCACTATAGNNNNANNNN CAGCGCACAGAGCAGCGGGACT  72 hTRBV7-9 TGTAATACGACTCACTATAGNNNNANNNN GAGATCCAGCGCACAGAGCAGG  73 hTRBV8-1 TGTAATACGACTCACTATAGNNNNANNNN CCCTCAACCCTGGAGTCTACTA  74 hTRBV8-2 TGTAATACGACTCACTATAGNNNNANNNN TCCCCAATCCTGGCATCCACCA  75 hTRBV9 TGTAATACGACTCACTATAGNNNNANNNN CTAAACCTGAGCTCTCTGGAGCT  76 hTRBV10-1 TGTAATACGACTCACTATAGNNNNANNNN CCCTCACTCTGGAGTCTGCTGC  77 hTRBV10-2 TGTAATACGACTCACTATAGNNNNANNNN CCCTCACTCTGGAGTCAGCTAC  78 hTRBV10-3 TGTAATACGACTCACTATAGNNNNANNNN TCCTCACTCTGGAGTCCGCTAC  79 hTRBV11-1 TGTAATACGACTCACTATAGNNNNANNNN CCACTCTCAAGATCCAGCCTGCA  80 hTRBV12-1 TGTAATACGACTCACTATAGNNNNANNNN GAGGATCCAGCCCATGGAACCCA  81 hTRBV12-2 TGTAATACGACTCACTATAGNNNNANNNN CTGAAGATCCAGCCTGCAGAGC  82 hTRBV12-3 TGTAATACGACTCACTATAGNNNNANNNN CAGCCCTCAGAACCCAGGGACT  83 hTRBV13 TGTAATACGACTCACTATAGNNNNANNNN GAGCTCCTTGGAGCTGGGGGACT  84 hTRBV14 TGTAATACGACTCACTATAGNNNNANNNN GGTGCAGCCTGCAGAACTGGAG  85 hTRBV15 TGTAATACGACTCACTATAGNNNNANNNN GACATCCGCTCACCAGGCCTGG  86 hTRBV16 TGTAATACGACTCACTATAGNNNNANNNN TGAGATCCAGGCTACGAAGCTT  87 hTRBV17 TGTAATACGACTCACTATAGNNNNANNNN GAAGATCCATCCCGCAGAGCCG  88 hTRBV18 TGTAATACGACTCACTATAGNNNNANNNN GGATCCAGCAGGTAGTGCGAGG  89 hTRBV19 TGTAATACGACTCACTATAGNNNNANNNN CACTGTGACATCGGCCCAAAAG  90 hTRBV20 TGTAATACGACTCACTATAGNNNNANNNN CTGACAGTGACCAGTGCCCATC  91 hTRBV21 TGTAATACGACTCACTATAGNNNNANNNN GAGATCCAGTCCACGGAGTCAG  92 hTRBV22 TGTAATACGACTCACTATAGNNNNANNNN GTGAAGTTGGCCCACACCAGCCA  93 hTRBV23 TGTAATACGACTCACTATAGNNNNANNNN CCTGGCAATCCTGTCCTCAGAA  94 hTRBV24 TGTAATACGACTCACTATAGNNNNANNNN GAGTCTGCCATCCCCAACCAGA  95 hTRBV25 TGTAATACGACTCACTATAGNNNNANNNN GGAGTCTGCCAGGCCCTCACA  96 hTRBV26 TGTAATACGACTCACTATAGNNNNANNNN GAAGTCTGCCAGCACCAACCAG  97 hTRBV27 TGTAATACGACTCACTATAGNNNNANNNN GGAGTCGCCCAGCCCCAACCAG  98 hTRBV28 TGTAATACGACTCACTATAGNNNNANNNN GGAGTCCGCCAGCACCAACCAG  99 hTRBV29 TGTAATACGACTCACTATAGNNNNANNNN GTGAGCAACATGAGCCCTGAAGA 100 hTRBV30 TGTAATACGACTCACTATAGNNNNANNNN GAGTTCTAAGAAGCTCCTTCTCA

TABLE 5 V segments targeted by each primer used for the amplification ofTCR β chain V segments. TCR b chain V SEQ ID NO segment name Targeted Vsegment(s) 51 hTRBV1 hTRBV01 52 hTRBV2 hTRBV02 53 hTRBV3-1 hTRBV03-1,hTRBV03-2 54 hTRBV4-1 hTRBV04-1 55 hTRBV4-2 hTRBV04-2, hTRBV04-3 56hTRBV5-1 hTRBV05-1 57 hTRBV5-2 hTRBV05-2 58 hTRBV5-3 hTRBV05-3 59hTRBV5-4 hTRBV05-4, hTRBV05-5, hTRBV05-6, hTRBV05-7, hTRBV05-8 60hTRBV6-1 hTRBV06-1 61 hTRBV6-2 hTRBV06-2, hTRBV06-3 62 hTRBV6-4hTRBV06-4 63 hTRBV6-5 hTRBV06-5 64 hTRBV6-6 hTRBV06-6, hTRBV06-9 65hTRBV6-7 hTRBV06-7 66 hTRBV6-8 hTRBV06-8 67 hTRBV7-1 hTRBV07-1 68hTRBV7-2 hTRBV07-2, hTRBV07-8 69 hTRBV7-3 hTRBV07-3, hTRBV07-4 70hTRBV7-5 hTRBV07-5 71 hTRBV7-6 hTRBV07-6, hTRBV07-7 72 hTRBV7-9hTRBV07-9 73 hTRBV8-1 hTRBV08-1 74 hTRBV8-2 hTRBV08-2 75 hTRBV9 hTRBV0976 hTRBV10-1 hTRBV10-1 77 hTRBV10-2 hTRBV10-2 78 hTRBV10-3 hTRBV10-3 79hTRBV11-1 hTRBV11-1, hTRBV11-2, hTRBV11-3 80 hTRBV12-1 hTRBV12-1 81hTRBV12-2 hTRBV12-2 82 hTRBV12-3 hTRBV12-3, hTRBV12-4, hTRBV12-5 83hTRBV13 hTRBV13 84 hTRBV14 hTRBV14 85 hTRBV15 hTRBV15 86 hTRBV16 hTRBV1687 hTRBV17 hTRBV17 88 hTRBV18 hTRBV18 89 hTRBV19 hTRBV19 90 hTRBV20hTRBV20 91 hTRBV21 hTRBV21 92 hTRBV22 hTRBV22 93 hTRBV23 hTRBV23 94hTRBV24 hTRBV24 95 hTRBV25 hTRBV25 96 hTRBV26 hTRBV26 97 hTRBV27 hTRBV2798 hTRBV28 hTRBV28 99 hTRBV29 hTRBV29 100 hTRBV30 hTRBV30

TABLE 6Primers for TCR gene amplification. Primer pair for sequencing of TCR αgenes: SEQ ID NO 101 and 102. Primer pair for sequencing of TCR  βgenes: SEQ ID NO 101 and 103. SEQ ID NO Primer name Primer sequenceTCR chain 101 Forward primer T7 TRAV/TRBV TGTAATACGACTCACTATAG α and β102 Reverse primer PCR 1 TRAV GGCCACAGCACTGTTGCTCTTGAAG α 103Reverse primer PCR 1 TRABV CCACTGTGCACCTCCTTCCCATTC β

TABLE 7 Reverse primers for TCR gene amplification thatdid not result in successful amplification of PCR products. SEQ ID NOPrimer sequence TCR chain 104 TCGACCAGCTTGACATCACAGG α 105CAGATTTGTTGCTCCAGGCCACAG α 106 TCTGTGATATACACATCAGAATC α 107GAATCAAAATCGGTGAATAGGCAG α 108 GGCAGACAGACTTGTCACTGGATT α 109TAGGACACCGAGGTAAAGCCAC β 110 CTGGGTGACGGGTTTGGCCCTAT β 111TTGACAGCGGAAGTGGTTGC β 112 GGCTGCTCAGGCAGTATCTGGAGTC β 113GCCAGGCACACCAGTGTGGCCTTTT β

TABLE 8Primers for addition of Next Generation Sequencing adapters. The primer portioncorresponding to the Illumina ®adapters (forward and reverse) is underlined inforward and reverse primers shown below. Primer pair for sequencing of TCR αgenes: SEQ ID NOS: 114 and 115. Primer pair for sequencing of TCR βgenes:  SEQ ID NOS: 114 and 116. SEQ ID TCR NO Primer namePrimer sequence chain 114 Forward primerAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT α and β Illumina_T7CTTCCGATCTTGTAATACGACTCACTATAG TRAV/TRBV 115 Reverse primerCAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC α PCR 2 TRACGTGTGCTCTTCCGATCCTCAGCTGGTACACGGCAGGGTCA 116 Reverse primerCAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC β PCR 2 TRBCGTGTGCTCTTCCGATCAAACACAGCGACCTCGGGTGGGAAC

Example 2: Exemplary Protocol for the SEQTR Method Using NexteraAdapters

TCR α and β chain genes were sequenced in two independent reactions.

-   -   1) Starting material and RNA extraction        -   To obtain sufficient amounts of RNA in the extraction, a            minimum of 500,000 T-cells were used as starting material.            Alternatively, and especially in instances where fewer            T-cells were available, T-cells were mixed with 50,000 mouse            3T3 cells that served as carrier. T-cell RNA was extracted            using the RNeasy® Micro Kit from Qiagen Inc. according the            manufacturer's instruction with the following modification:            Elution was performed with 20 μl of water preheated to            50° C. RNA quality and quantity was verified using a            fragment analyzer.    -   2) cRNA synthesis by in vitro transcription (IVT):        -   In vitro transcription of isolated RNA was performed using            the MessageAmp™ II aRNA Amplification Kit from Ambion®            (Thermo Fisher Scientific), which contains enzymes, buffers            and nucleotides required to perform the first and second            strand cDNA and the in vitro transcription. The kit also            provides all columns and reagents needed for the cDNA and            cRNA purifications. RNA amplification was performed            according to the manufacturer's instructions with the            following modifications: 1) Between 0.5 and 1 μg of total            RNA as was used as starting material. 2) The IVT was            performed in a final volume of 40 μl, and incubated at            37° C. for 16 h. Purified cRNA was quantified by absorbance            using a NanoDrop™ spectrophotometer (Thermo Fisher            Scientific).    -   3) cDNA synthesis by reverse transcription:        -   The reverse transcription of the cRNA was performed with the            SuperScript® III from Invitrogen (Thermo Fisher Scientific).            The kit provides the enzyme, the buffer and the            dithiothreitol (DTT) needed for the reaction.            Deoxynucleotides (dNTPs) and RNAsin® Ribonuclease inhibitor            were purchased from Promega. The sequences for the primers            used for the reverse transcription can be found in Table 9            (primers for sequencing TCR α chain genes) and Table 10            (primers for sequencing TCR β chain genes).        -   500 ng of cRNA were used as starting material for the            reverse transcription. cRNA was mixed with 1 μl hTRAV or            hTRBV primers mix (2 μM each) and 1 μl dNTP (25 mM) in a            final volume of 13 μl. The mix was first incubated at 70° C.            for 10 min, then at 50° C. for 30 s. 4 μl 5× buffer, 1 μl            DTT (100 mM), 1 μl SuperScript III and 1 μl RNAsin® were            added to the mix. The samples were subsequently incubated            for at 55° C. 1 h and then at 85° C. for 5 min. After the            cDNA synthesis, 1 μg DNase-free RNase (Roche) was added to            the cDNA and incubated at 37° C. for 30 min to remove the            cRNA.    -   4) TCR gene amplification:        -   TCR gene amplification was performed using a Phusion®            High-Fidelity DNA polymerase (New England Biolabs) under the            following conditions:        -   PCR mix: 1 μl cDNA from step 3, 0.4 μl dNTPs (10 mM), 0.4 μl            primer mix (20 μM Nextera5′, 10 μM Reverse primer PCR 1 TRAV            or 2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5×            buffer and 0.2 μl Phusion® enzyme in a total volume of 10            μl.        -   PCR conditions:            -   94° C. for 5 min            -   20 cycles of                -   98° C. for 10 s                -   55° C. for 30 s                -   72° C. for 30 s            -   72° C. for 2 min        -   PCR products were purified using 1 μl of ExoSAP-IT® PCR            Product Cleanup Kit (Affymetrix) according to the            manufacturer's instructions.    -   5) Addition of Next Generation Sequencing adapters:        -   ILLUMINA® sequencing adapters were added by PCR using a            Phusion® High-Fidelity DNA polymerase (New England Biolabs).            The following mix was added to the 11 μl of PCR1: 1 μl dNTPs            (10 mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl            5× buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.        -   PCR conditions:            -   94° C. for 5 min            -   perform 25 cycles of:                -   98° C. for 10 s                -   55° C. for 30 s                -   72° C. for 30 s            -   72° C. for 2 min    -   6) TCR library purification:        -   10 μl of the PCR product from step 5 were purified using an            AMPURE XP beads (Beckman Coulter) according to the            manufacturer's instruction. Samples could then directly be            used for ILLUMINA® sequencing.

TABLE 9 Preferred primer sequences for amplification of TCR αchain V segments. N can beany nucleotide. The sequences for primers presented in this table consist ofthree parts (listed from 5′ to 3′): T7 adapter, barcode and TCR αchain V segment. SEQ Sequence Nextera Sequence ID Primeradapter portion  barcode portion Sequence TCR α chain V segment NO nameof the primer of the primer portion of the primer 261 hTRAV1-1TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTCTACAGGAGCTCCAGATGAAAG GTATAAGAGACAG262 hTRAV1-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTTTGAAGGAGCTCCAGATGAAAGGTATAAGAGACAG 263 hTRAV2 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTGCTCATCCTCCAGGTGCGGGA GTATAAGAGACAG 264 hTRAV3 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GAAGAAACCATCTGCCCTTGTGA GTATAAGAGACAG 265 hTRAV4TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGCCCCGGGTTTCCCTGAGCGAC GTATAAGAGACAG266 hTRAV5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCGCATTGCAGACACCCAGTATAAGAGACAG 267 hTRAV6 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTTGTTTCATATCACAGCCTCCCA GTATAAGAGACAG 268 hTRAV7 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GCTTGTACATTACAGCCGTGCA GTATAAGAGACAG 269 hTRAV8-1/8-3TCGTCGGCAGCGTCAGATGT HHHHHNNNN ATCTGAGGAAACCCTCTGTGCA GTATAAGAGACAG 270hTRAV8-2/8-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTGACGAAACCCTCAGCCCATGTATAAGAGACAG 271 hTRAV8-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNNCCTATGCCTGTCTTTACTTTAATC GTATAAGAGACAG 272 hTRAV8-6 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CTTGAGGAAACCCTCAGTCCATAT GTATAAGAGACAG 273 hTRAV8-7TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAACCATCAACCCATGTGAGTGA GTATAAGAGACAG274 hTRAV9-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTTGGAGAAAGACTCAGTTCAAGTATAAGAGACAG 275 hTRAV9-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNNACTTGGAGAAAGGCTCAGTTCAA GTATAAGAGACAG 276 hTRAV10 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CTGCACATCACAGCCTCCCA GTATAAGAGACAG 277 hTRAV11TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTTGGAATATCGCAGCCTCTCAT GTATAAGAGACAG278 hTRAV12-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTGCTCATCAGAGACTCCAAGGTATAAGAGACAG 279 hTRAV12-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNNCTCTGCTCATCAGAGACTCCCAG GTATAAGAGACAG 280 hTRAV12-3 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CCTTGTTCATCAGAGACTCACAG GTATAAGAGACAG 281 hTRAV13-1TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCACATCACAGAGACCCAA GTATAAGAGACAG282 hTRAV13-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCAAATTGCAGCTACTCAAGTATAAGAGACAG 283 hTRAV14 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTTGTCATCTCCGCTTCACAACTGG GTATAAGAGACAG 284 hTRAV15 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GTTTTGAATATGCTGGTCTCTCAT GTATAAGAGACAG 285 hTRAV16TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGAAGAAACCATTTGCTCAAGA GTATAAGAGACAG286 hTRAV17 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTTGTTGATCACGGCTTCCCGGGTATAAGAGACAG 287 hTRAV18 TCGTCGGCAGCGTCAGATGT HHHHHNNNNACCTGGAGAAGCCCTCGGTGCA GTATAAGAGACAG 288 hTRAV19 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CACCATCACAGCCTCACAAGTCGT GTATAAGAGACAG 289 hTRAV20TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTTCTGCACATCACAGCCCCTA GTATAAGAGACAG 290hTRAV21 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTTATACATTGCAGCTTCTCAGCCGTATAAGAGACAG 291 hTRAV22 TCGTCGGCAGCGTCAGATGT HHHHHNNNNGTACATTTCCTCTTCCCAGACCAC GTATAAGAGACAG 292 hTRAV23 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CATTGCATATCATGGATTCCCAGC GTATAAGAGACAG 293 hTRAV24TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTATTTGTACATCAAAGGATCCC GTATAAGAGACAG294 hTRAV25 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCTCCCTGCACATCACAGCCAGTATAAGAGACAG 295 hTRAV26-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTTGATCCTGCCCCACGCTACGCTGA GTATAAGAGACAG 296 hTRAV26-2TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGATCCTGCACCGTGCTACCTTGA GTATAAGAGACAG297 hTRAV27 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTCTCTCCACATCACTGCAGCCGTATAAGAGACAG 298 hTRAV28 TCGTCGGCAGCGTCAGATGT HHHHHNNNNGCCACCTATACATCAGATTCCCA GTATAAGAGACAG 299 hTRAV29 TCGTCGGCAGCGTCAGATGTHHHHHNNNN TCTCTGCACATTGTGCCCTCCCA GTATAAGAGACAG 300 hTRAV30TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTGTACCTTACGGCCTCCCAGCT GTATAAGAGACAG301 hTRAV31 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTATCATATCATCATCACAGCCAGTATAAGAGACAG 302 hTRAV32 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTCCCTGCATATTACAGCCACCCAA GTATAAGAGACAG 303 hTRAV33 TCGTCGGCAGCGTCAGATGTHHHHHNNNN ACCTCACCATCAATTCCTTAAAAC GTATAAGAGACAG 304 hTRAV34TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCATATCACAGCCTCCCAG GTATAAGAGACAG305 hTRAV35 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTCCTGAATATCTCAGCATCCATGTATAAGAGACAG 306 hTRAV36 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTCCTGAACATCACAGCCACCCAG GTATAAGAGACAG 307 hTRAV37 TCGTCGGCAGCGTCAGATGTHHHHHNNNN TCCCTGCACATACAGGATTCCCAG GTATAAGAGACAG 308 hTRAV38TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAAGATCTCAGACTCACAGCTGG GTATAAGAGACAG 309hTRAV39 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCGTCTCAGCACCCTCCACATCAGTATAAGAGACAG 310 hTRAV40 TCGTCGGCAGCGTCAGATGT HHHHHNNNNCCATTGTGAAATATTCAGTCCAGG GTATAAGAGACAG

TABLE 10 Preferred primer sequences for amplification of TCR βchain V segments. N can beany nucleotide. The sequences for primers presented in this table consist of threeparts (listed from 5′ to 3′): T7 adapter, barcode and TCR βchain V segment. SEQ Sequence T7 Sequence ID Primer adapter portion barcode portion Sequence TCR β chain V segment NO name of the primerof the primer portion of the primer 311 hTRBV1 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GTGGTCGCACTGCAGCAAGAAGA GTATAAGAGACAG 312 hTRBV2TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATCCGGTCCACAAAGCTGGAGGA GTATAAGAGACAG313 hTRBV3-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CATCAATTCCCTGGAGCTTGGTGAGTATAAGAGACAG 314 hTRBV4-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTTCACCTACACGCCCTGCAGCCAG GTATAAGAGACAG 315 hTRBV4-2 TCGTCGGCAGCGTCAGATGTHHHHHNNNN TTCACCTACACACCCTGCAGCCAG GTATAAGAGACAG 316 hTRBV5-1TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAATGTGAGCACCTTGGAGCTGG GTATAAGAGACAG 317hTRBV5-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TACTGAGTCAAACACGGAGCTAGGGTATAAGAGACAG 318 hTRBV5-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNNGCTCTGAGATGAATGTGAGTGCCT GTATAAGAGACAG 319 hTRBV5-4 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CTGAGCTGAATGTGAACGCCTT GTATAAGAGACAG 320 hTRBV6-1TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTTCTCGCTCAGGCTGGAGT GTATAAGAGACAG 321hTRBV6-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGGGGTTGGAGTCGGCTGCTCGTATAAGAGACAG 322 hTRBV6-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNNCCCCTCACGTTGGCGTCTGCTG GTATAAGAGACAG 323 hTRBV6-5 TCGTCGGCAGCGTCAGATGTHHHHHNNNN TCCCGCTCAGGCTGCTGTCGGC GTATAAGAGACAG 324 hTRBV6-6TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATTTCCCGCTCAGGCTGGAGT GTATAAGAGACAG 325hTRBV6-7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCCCTCAAGCTGGAGTCAGCTGTATAAGAGACAG 326 hTRBV6-8 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTCCCACTCAGGCTGGTGTCGGC GTATAAGAGACAG 327 hTRBV7-1 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CTCTGAAGTTCCAGCGCACACA GTATAAGAGACAG 328 hTRBV7-2TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATCCAGCGCACACAGCAGGAG GTATAAGAGACAG 329hTRBV7-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTCTGAAGATCCAGCGCACAGAGTATAAGAGACAG 330 hTRBV7-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNNAGATCCAGCGCACAGAGCAAGG GTATAAGAGACAG 331 hTRBV7-6 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CAGCGCACAGAGCAGCGGGACT GTATAAGAGACAG 332 hTRBV7-9TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGATCCAGCGCACAGAGCAGG GTATAAGAGACAG 333hTRBV8-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCAACCCTGGAGTCTACTAGTATAAGAGACAG 334 hTRBV8-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTCCCCAATCCTGGCATCCACCA GTATAAGAGACAG 335 hTRBV9 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CTAAACCTGAGCTCTCTGGAGCT GTATAAGAGACAG 336 hTRBV10-1TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCACTCTGGAGTCTGCTGC GTATAAGAGACAG 337hTRBV10-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCACTCTGGAGTCAGCTACGTATAAGAGACAG 338 hTRBV10-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTCCTCACTCTGGAGTCCGCTAC GTATAAGAGACAG 339 hTRBV11-1 TCGTCGGCAGCGTCAGATGTHHHHHNNNN CCACTCTCAAGATCCAGCCTGCA GTATAAGAGACAG 340 hTRBV12-1TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGGATCCAGCCCATGGAACCCA GTATAAGAGACAG 341hTRBV12-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGAAGATCCAGCCTGCAGAGCGTATAAGAGACAG 342 hTRBV12-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNNCAGCCCTCAGAACCCAGGGACT GTATAAGAGACAG 343 hTRBV13 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GAGCTCCTTGGAGCTGGGGGACT GTATAAGAGACAG 344 hTRBV14TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGTGCAGCCTGCAGAACTGGAG GTATAAGAGACAG 345hTRBV15 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GACATCCGCTCACCAGGCCTGGGTATAAGAGACAG 346 hTRBV16 TCGTCGGCAGCGTCAGATGT HHHHHNNNNTGAGATCCAGGCTACGAAGCTT GTATAAGAGACAG 347 hTRBV17 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GAAGATCCATCCCGCAGAGCCG GTATAAGAGACAG 348 hTRBV18TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGATCCAGCAGGTAGTGCGAGG GTATAAGAGACAG 349hTRBV19 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CACTGTGACATCGGCCCAAAAGGTATAAGAGACAG 350 hTRBV20 TCGTCGGCAGCGTCAGATGT HHHHHNNNNCTGACAGTGACCAGTGCCCATC GTATAAGAGACAG 351 hTRBV21 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GAGATCCAGTCCACGGAGTCAG GTATAAGAGACAG 352 hTRBV22TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGAAGTTGGCCCACACCAGCCA GTATAAGAGACAG 353hTRBV23 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGGCAATCCTGTCCTCAGAAGTATAAGAGACAG 354 hTRBV24 TCGTCGGCAGCGTCAGATGT HHHHHNNNNGAGTCTGCCATCCCCAACCAGA GTATAAGAGACAG 355 hTRBV25 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GGAGTCTGCCAGGCCCTCACA GTATAAGAGACAG 356 hTRBV26TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGTCTGCCAGCACCAACCAG GTATAAGAGACAG 357hTRBV27 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCGCCCAGCCCCAACCAGGTATAAGAGACAG 358 hTRBV28 TCGTCGGCAGCGTCAGATGT HHHHHNNNNGGAGTCCGCCAGCACCAACCAG GTATAAGAGACAG 359 hTRBV29 TCGTCGGCAGCGTCAGATGTHHHHHNNNN GTGAGCAACATGAGCCCTGAAGA GTATAAGAGACAG 360 hTRBV30TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTTCTAAGAAGCTCCTTCTCA GTATAAGAGACAG

TABLE 11Primers for TCR gene amplification. Primer pair for sequencing of TCR  αgenes: SEQ ID NOs: 256 and 257. Primer pair for sequencing of TCR βgenes: SEQ ID NOs: 256 and 258. The primer portion corresponding to the Illumina ® adapters (forward and reverse) is underlined in reverseprimers shown below. SEQ ID NO Primer name Primer sequence TCR chain 256Forward primer Nextera 5′ TCGTCGGCAGCGTC α and β 257Reverse primer PCR 1 TRAV GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAATCAAAATCGGTGAATA α GGCAG 258 Reverse primer PCR 1 TRBVGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAGGCCAGGCACACCAGTGTGG β CCTTTT

TABLE 12 Primers used to add the full Nextera sequence to both TCRαand TCRβ. SEQ ID TCR NO Primer name Primer sequence chain 259Index Read 1 CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTC α and β 260Index Read 2 AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCG α

Example 3: Exemplary Protocol for the SEQTR Method without In VitroTranscription

TCR α and β chain genes were sequenced in two independent reactions.

-   -   1) Starting material and RNA extraction        -   To obtain sufficient amounts of RNA in the extraction, a            minimum of 500,000 T-cells were used as starting material.            Alternatively, and especially in instances where fewer            T-cells were available, T-cells were mixed with 50,000 mouse            3T3 cells that served as carrier. T-cell RNA was extracted            using the RNeasy® Micro Kit from Qiagen Inc. according the            manufacturer's instruction with the following modification:            Elution was performed with 20 μl of water preheated to            50° C. RNA quality and quantity was verified using a            fragment analyzer.    -   2) cDNA synthesis by reverse transcription:        -   The reverse transcription of the RNA was performed with the            SuperScript® III from Invitrogen (Thermo Fisher Scientific)            and oligo d(T). The kit provides the enzyme, the buffer and            the dithiothreitol (DTT) needed for the reaction.            Deoxynucleotides (dNTPs), oligo d(T) and RNAsin®            Ribonuclease inhibitor were purchased from Promega. 500 ng            of RNA were used as starting material for the reverse            transcription. RNA was mixed with 1 μl of oligo d(T) and 1            μl dNTP (25 mM) in a final volume of 13 μl. The mix was            first incubated at 70° C. for 10 min, then at 50° C. for            30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μl SuperScript            III and 1 μl RNAsin® were added to the mix. The samples were            subsequently incubated for at 55° C. 1 h and then at 85° C.            for 5 min.    -   3) Second strand cDNA synthesis:        -   cDNA was then used to synthesize the second strand,            performed using the Phusion® High-Fidelity DNA polymerase            (New England Biolabs) under the following conditions:        -   Mix: 20 μl cDNA from step 2, 4 μl dNTPs (10 mM), 2 μl TRAV            primer mix (Table 9), 2 μl TRBV primers mix (Table 10), 20            μl 5× buffer, 1 μl Phusion® enzyme in a total volume of 100            μl.        -   Synthesis conditions:            -   98° C. for 5 min            -   40° C. for 30 s            -   72° C. for 5 min    -   4) cDNA purification:        -   100 μl of the cDNA product from step 3 were purified using            an AMPURE XP beads (Beckman Coulter) according to the            manufacturer's instruction.    -   5) TCR gene amplification:        -   TCR gene amplification was performed using a Phusion®            High-Fidelity DNA polymerase (New England Biolabs) under the            following conditions:        -   PCR mix: 7 μl cDNA from step 4, 0.4 μl dNTPs (10 mM), 0.4 μl            primer mix (20 μM Nextera5′, 10 μM Reverse primer PCR 1 TRAV            or 2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5×            buffer and 0.2 μl Phusion® enzyme in a total volume of 10            μl.        -   PCR conditions:            -   94° C. for 5 min            -   20 cycles of                -   98° C. for 10 s                -   55° C. for 30 s                -   72° C. for 30 s            -   72° C. for 2 min        -   PCR products were purified using 1 μl of ExoSAP-IT® PCR            Product Cleanup Kit (Affymetrix) according to the            manufacturer's instructions.    -   6) Addition of Next Generation Sequencing adapters:        -   ILLUMINA® sequencing adapters were added by PCR using a            Phusion® High-Fidelity DNA polymerase (New England Biolabs).            The following mix was added to the 11 μl of PCR1: 1 μl dNTPs            (10 mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl            5× buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.        -   PCR conditions:            -   94° C. for 5 min            -   perform 25 cycles of:                -   98° C. for 10 s                -   55° C. for 30 s                -   72° C. for 30 s            -   72° C. for 2 min    -   7) TCR library purification:        -   10 μl of the PCR product from step 5 were purified using an            AMPURE XP beads (Beckman Coulter) according to the            manufacturer's instruction. Samples could then directly be            used for ILLUMINA® sequencing.

Example 4: Sensitivity of the TCR Sequencing Method

One of the challenges of TCR sequencing are the small amounts of geneticmaterial for each T-cell clone. In many cases, the number of T-cellsthat can be recovered from a given experiment is too small forresearchers to directly extract sufficient amounts of RNA for asubsequent amplification of the TCR genes. In such instances, theT-cells of interest can be mixed with 3T3 mouse cells, which serve as acarrier.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflexover ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of eachmixture was isolated and subjected to steps 2 to 4 of the SEQTR methodoutlined above (see Detailed Description of the Invention). PCR productswere separated on an agarose gel and visualized.

No TCR-specific PCR products were observed in samples that onlycontained 3T3 cells (see FIG. 4). However, TCR-specific bands weredetected in all other samples: Increasing amounts of CD8 positiveT-cells in the samples were correlated with increasing amounts ofTCR-specific PCR products and decreasing intensity of the unspecificdimer primer band. These data demonstrate that the SEQTR method issensitive enough to amplify TCR genes from as little as 1,000 T-cells,with no detectable background signal from the 3T3 carrier cells.

Example 5: Specificity of the SEQTR Method

Another challenge of TCR sequencing is the lack of specificamplification of TCR genes from complex samples. Competing TCRsequencing technologies such as services offered by AdaptiveBiotechnology are characterized by up to 90% unspecific amplification.As a result, only as little as 10% of all sequencing data areinformative for TCR repertoire determination, increasing cost andduration of any project aiming to sequence TCR repertoires.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflexover ( )}3 or 0 CD8 positive T-cells, respectively. TCR repertoires forthe individual samples were sequenced using the SEQTR method, and thepercentage of reads that corresponded to TCR or non-TCR sequences,respectively, was determined. As shown in FIG. 5, 93-97% of allsequencing reads indeed corresponded to TCR genes, independent of theamount of T-cells used as starting material. In summary, these data showthat TCR amplification using the SEQTR method is highly specific evenwhen as little as 1,000 T-cells are used as starting material.

Example 6: Unambiguous Identification of TCR Genes

In humans, the TCR locus comprises 54 different V segments for the TCR αchain and 65 different V segments for the TCR β chain. However, many ofthese V segments are highly homologous. Consequently, one of the bigchallenges of TCR sequencing is to successfully differentiate betweentwo or more TCR gene segments with high degrees of homology. Forinstance, depending on the choice of primer used in the amplification ofthe TCR gene and the length of the generated PCR product, the resultingsequencing data might be compatible with more than one V or J segment(in other words, two or more TCR V or J segments show 100% homology inthe sequenced region). In these cases, the TCR gene for a specific readcannot be unambiguously assigned/identified.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflexover ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of eachmixture was isolated and subjected to the TCR sequencing method. Out ofall the sequencing reads that were identified as TCR genes, it wasassessed if the V or J segments could be identified unambiguously. Thedata show that between 95% and 97% of all TCR sequencing reads could beassigned to a specific TCR segment, even when using as little as 1,000T-cells as genetic starting material (see FIG. 6). In summary, the datademonstrate the robustness of the SEQTR method as 90 to 93% of all readscan be used to identify TCR sequences once unspecific sequences andambiguous TCR sequences have been removed.

Due to the homology between V segments, it can be sometimes difficult toclearly identify the TCR sequence. hTRBV6-2 and hTRBV6-3 cannot bedifferentiated as they have 100% homology and thus will code for thesame TCR. Due to their sequences, hTRBV12-3 and hTRBV12-4 cannot bedifferentiated with the method disclosed herein. Only paired-endsequencing that will catch the 5′-end of the V segment can discriminatethese two sequences. Thus the hTRBV12-3 and hTRBV12-4 were considered asa unique sequence for the analysis of the repertoire.

Example 7: Linearity of TCR Gene Amplification

Because non-linear amplification of individual TCR sequences can lead toan incorrect over- or underrepresentation of the affected TCR genes inthe final TCR repertoire, linearity of amplification is a criticaldeterminant of the reliability and quality of the TCR sequencing data.

To test linearity of TCR gene amplification in our system, a fixedamount of DNA encoding a known TCR sequence was diluted at differentconcentrations into a DNA pool representing a naïve CD8 repertoire.Subsequently, the TCR repertoire of each sample was analyzed with SEQTR.

The observed frequency of the known TCR sequence in the entire TCRrepertoire was then sequenced for each dilution and compared to theexpected frequency. The scatter plot in FIG. 7 shows an excellentcorrelation (R²=0.99) between the dilution and the frequency of theknown TCR sequence in the repertoire observed after sequencing. Thesedata confirm the linearity of the amplification and suggest that resultsobtained using the SEQTR technique are quantitative.

Example 8: Reproducibility of the SEQTR Method

The reproducibility of the method was tested by performing twoindependent technical replicates starting from the same sample. Thefrequencies for each V-J rearrangement in the TCR β chains weredetermined and compared between the two replicates, as illustrated inFIG. 8. Each sphere represents a single V-J rearrangement that wasdetected in both replicates. Each sphere represents a single V-Jrearrangement with the size of a sphere indicating the relativefrequency of the specific V-J recombination. Grey spheres representrearrangements for which the relative frequencies detected in the tworeplicates differed by less than two-fold. Black spheres representrearrangements for which the relative frequencies detected in the tworeplicates differed by more than two-fold. Consistent with commonpractice in the analysis of gene expression data, differences betweenreplaces of less than 2-fold are not considered significant.

The data show that only 13% of all V-J rearrangements showed asignificant frequency difference of more than two-fold between the twotechnical replicates (see FIG. 8 upper inset). However, as illustratedin FIG. 8, V-J recombinations that were significantly different betweenthe technical replicates were rather poorly expressed, as indicated bythe small sizes of the black spheres. Therefore, if the frequencies ofthe individual V-J rearrangement are taken into consideration, only 0.5%of the sequences showed more than a two-fold difference between thereplicates (see FIG. 8 lower inset), demonstrating that the SEQTR methodis very reproducible.

Example 9: Sequencing of Example Repertoires Using the SEQTR Method

The SEQTR method was tested on three different type of CD8 positiveT-cells:

-   -   (1) T-cell population 1: CD8 positive T-cells isolated from        peripheral blood mononuclear cells (PBMCs).    -   (2) T-cell population 2: CD8 positive T-cells as in population 1        were FACS sorted using tetramers. Tetramers are MHC molecules        presenting a specific peptide, linked to fluorescent dye.        Tetramers bind T-cell expressing a TCR that specifically        recognizes the peptide. The fluorescent dye allows sorting of        the desired T-cells by FACS.    -   (3) T-cell population 3: CD8 positive T-cells as in population 2        that were subsequently expanded in vitro.

The relative frequencies of each V-J rearrangement were determined usingthe SEQTR method (see FIG. 9). As expected, the naïve TCR repertoirederived from PBMC (population 1) is highly diverse (see FIG. 9A). Almostall the possible V-J rearrangements are represented in the sample, withno single V-J rearrangement exhibiting a frequency of over 11% Therepertoire of the tetramer sorted CD8 positive T-cell subset 2 (see FIG.9B) is less diverse as compared to the naïve one. Not only are fewer V-Jrearrangements present in the repertoire overall. Moreover, two V-Jrearrangements are clearly dominant, exhibiting frequencies of over 20%.These V-J rearrangements represent the few T-cells that recognize theepitope TEDYMIHII (SEQ ID NO: 236) conjugated to the tetramer and thatwere enriched during the tetramer purification step. Finally, the rapidclonal expansion (population 3) of the tetramer-purified T-cellsenhances the bias of the TCR repertoire towards the T-cell clonesalready dominating subset 2. Consequently, part of the low frequency V-Jrearrangements are lost and not detected anymore (see FIG. 9C). Insummary, these data illustrate that the SEQTR method is well suited todifferentiate between TCR repertoires with different degrees ofdiversities.

Example 10: Comparison of the SEQTR Method with Low-Throughput SingleCell Cloning

In order to determine how accurate the TCR repertoire data obtainedusing the SEQTR method were as compared to the true TCR repertoirepresent in a given T-cell population, we compared our results to dataobtained by single cell sequencing.

Tetramer-specific CD8 were sorted from PBMC by FACS. The recovered cellpopulation was split in two. Half of the cells were subjected to theSEQTR method to sequence the TCR repertoire. For the other half of thecells, individual T-cell clones were isolated and expanded in vitro(single cell cloning). Once the clones were established, the TCR genesof each T-cell clones were amplified and sequenced using classicalSanger sequencing (see FIG. 10A).

Among the 42 individual clones tested using the single cell method, sixdifferent TCRs were identified (see FIG. 10B). Using the SEQTR method,116 different TCR genes were found (the eight most frequently observedV-J rearrangements are shown in FIG. 10C, also see Table 13 and Table14). Indeed, the five TCRs most frequently observed with the single cellcloning technique also correspond to the five clones most frequentlyobserved when applying the SEQTR method. Overall, all six TCR clonesidentified with single cell sequencing are represented among the eightTCRs with the highest frequencies observed in the SEQTR method. Insummary, these data suggest that the SEQTR method produces a truerepresentation of the actual TCR repertoire of a given T-cellpopulation.

TABLE 13 CDR3 regions of TCR clones identified using thesingle cell sequencing method. SEQ ID NO CDR3 region 237CASSRHVGGVPEAFFG 238 CASSIGRGSEQYFG 239 CASSDVLSGEAFFG 240CASQGHKNTEAFFG 241 CASSLGPGGVKTNEKLFFG 242 CASSLGPGGVKTNEKLFFG

TABLE 14 Eight most frequently observed V-J rearrangementsof the 116 different TCR genes identified using the SEQTR method.SEQ ID NO CDR3 region 243 CASSDVLSGEAFFG 244 CASQGHKNTEAFFG 245CASSLGPGGVKTNEKLFFG 246 CASSIGRGSEQYFG 247 CASSRHVGGVPEAFFG 248CASSASKGQPQHFG 249 CASQGHKNTEAFFG 250 CASSLGPGGVKTNEKLFFG

Example 11: TCR Sequencing Services Offered by Adaptive BiotechnologyProvide Sequencing Data that May Reflect Up to 90% Unspecific TCRAmplification

Tumor samples from 16 patients were collected and the tumor cells wereseparated from the surrounding tissue (stroma). In addition,epitope-specific TIL were sorted by FACS from the tumor samples usingtetramer staining (TET). Finally, the tumor cells were engrafted intohumanized mice. After some time, the tumor was collected and epitopespecific TIL were sorted by FACS.

DNA extraction was performed for each sample. DNA was sent to AdaptiveBiotechnology for TCR sequencing (immunoSEQ® method, survey protocol200,000-300,000 reads per sample).

In 80% of the samples, the immunoSEQ® method failed to generate 200,000reads per samples, suggesting that the immunoSEQ® method fails togenerate TCR repertoires with significant reliability.

Example 12: Amplification of TCR Genes from PBMC and CD4 PositiveT-Cells

RNA was isolated from 10{circumflex over ( )}6 PBMC or 10{circumflexover ( )}6 CD4 positive T-cells, respectively, from three independentsamples, The RNA was then subjected to steps 2 to 4 of the SEQTR methodoutlined above (see Detailed Description of the Invention). PCR productswere separated on an agarose gel and visualized (see FIG. 12). OnlyTCR-specific bands are observed, suggesting that the SEQTR method cannotonly be used for CD8 positive T-cells (see Example 7), but also for CD4positive T-cells and even for T-cells that are part of a complex mixtureof other PBMCs.

The foregoing examples and description of the embodiments should betaken as illustrating, rather than as limiting the present invention asdefined by the claims. As will be readily appreciated, numerousvariations and combinations of the features set forth above can beutilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference herein in their entireties.

As described and claimed herein, including in the accompanying drawings,reference is made to particular features, including method steps. It isto be understood that the disclosure of the invention in thisspecification includes all possible combinations of such particularfeatures. For example, where a particular feature is disclosed in thecontext of a particular aspect or embodiment, or a particular claim,that feature can also be used, to the extent possible, in combinationwith and/or in the context of other particular aspects and embodiments,and in the disclosed methods, systems and kits generally.

Where reference is made herein to a method comprising two or moredefined steps, the defined steps can be carried out in any order orsimultaneously (except where the context excludes that possibility), andthe method can include one or more other steps which are carried outbefore any of the defined steps, between two of the defined steps, orafter all the defined steps (except where the context excludes thatpossibility).

TABLE 15 TCR αchain V segments and binding sites for primers presented in Table 2 and Table 9.The sequence for each V segment presented in this table consists of three parts (listedfrom 5′to 3′): Sequence upstream of primer binding site, sequence of the primer bindingsite, sequence downstream of the primer binding site. V SEQ PrimerhTRAV sequence segment Primer ID binding site downstream of name name NOhTRAV sequence upstream of primer binding site within hTRAVprimer binding site hTRAV01-1 hTRAV01-1 117ATGTGGGGAGCTTTCCTTCTCTATGTTTCCATGAAGATGGGAGGCACTGCAGGACAAAGCCTTGAGCAGCCCTCTCTTCTACAG ACTCTGCCTCTTAGAAGTGACAGCTGTGGAAGGAGCCATTGTCCAGATAAACTGCACGTACCAGACATCTGGGTTTTATGGGCTGTCGAGCTCCAG CTTCTGCGCTGTCTGGTACCAGCAACATGATGGCGGAGCACCCACATTTCTTTCTTACAATGCTCTGGATGGTTTGGAGGAGACAGGATGAAAG GAGAGA TCGTTTTTCTTCATTCCTTAGTCGCTCTGATAGTTATGGTTACCTC hTRAV01-2hTRAV01-2 118ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACTACAGGACAAAACATTGACCAGCCCACTCTTTTGAAG ACTCTGCCTCTTAGAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGGCTGTTGAGCTCCAG CCTCTGTGCTGTCTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCTTACAATGTTCTGGATGGTTTGGAGGAGAAAGGATGAAAG GAGAGA TCGTTTTTCTTCATTCCTTAGTCGGTCTAAAGGGTACAGTTACCTC hTRAV02hTRAV02 119ATGGCTTTGCAGAGCACTCTGGGGGCGGTGTGGCTAGGGCTTCTCCTCAACTCTCTCTGGAAGGTTGCAGAAAGCTGCTCATCC GGCAGATGCTGCAAGGACCAAGTGTTTCAGCCTTCCACAGTGGCATCTTCAGAGGGAGCTGTGGTGGAAATCTTCTGTAATCACTCTTCCAGGTGC TGTTTACTACTGTGTGTCCAATGCTTACAACTTCTTCTGGTACCTTCACTTCCCGGGATGTGCACCAAGACTCCTTGTTAAAGGCTCAAGGGA GCTGTGGAGGA AGCCTTCTCAGCAGGGACGATACAACATGACCTATGAACGGTTCTCTTCATCGChTRAV03 hTRAV03 120ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCACATTGAGTGGGCTGAGAGCTCAGTCAGTGGCTCAGGAAGAAACC GCGACTCCGCTTCCGGAAGATCAGGTCAACGTTGCTGAAGGGAATCCTCTGACTGTGAAATGCACCTATTCAGTCTCTGGAAACCCTATCTGCCCT TGTACTTCTGTGCTATCTTTTTTGGTATGTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGAAATACATCACAGGGGATAACCTGGTGTGA TGTGAGAGACATTAAAGGCAGCTATGGCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCACCT hTRAV04hTRAV04 121ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTGAGCCTTGCTAAGACCACCCAGCCCATCCCTGCCCCG ACTGCTGTGTACTCCATGGACTCATATGAAGGACAAGAAGTGAACATAACCTGTAGCCACAACAACATTGCTACAAATGATTATATGGTTTCCCT TACTGCCTCGTGCACGTGGTACCAACAGTTTCCCAGCCAAGGACCACGATTTATTATTCAAGGATACAAGACAAAAGTTACAAACGGAGCGAC GGTGACA AAGTGGCCTCCCTGTTTATCCCTGCCGACAGAAAGTCCAGCACTCTGAGhTRAV05 hTRAV05 122ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTGTATGAGTAGAGGAGAGGATGTGTCTCTGCGC GACTGGGGACTCGAGCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCATTGCAGAC AGCTATCTACTTACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACACCCA CTGTGCAGAGAGATGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTG TA hTRAV06hTRAV06 123ATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTGGACTGGGTGAAGAGCCAAAAGATAGAATTGTTTCAT GCCTGCAGACTCCAGAATTCCGAGGCCCTGAACATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTATTCCCCATCACAGCC AGCTACCTACCTAGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCTGTTTTCTTGCTACTCATACGTGAAAATGAGATCCCA CTGTGCTCTAGAAAGAAAAAAGGAAAGAAAGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGT CA hTRAV07hTRAV07 124ATGGAGAAGATGCGGAGACCTGTCCTAATTATATTTTGTCTATGTCTTGGCTGGGCAAATGGAGAAAACCAGGTGGCTTGTACA GCCTGAAGATTCGAGCACAGCCCTCATTTTCTGGGACCCCAGCAGGGAGACGTTGCCTCCATGAGCTGCACGTACTCTGTCAGTCGTTTACAGCCG AGCCACCTATTTTTTAACAATTTGCAGTGGTACAGGCAAAATACAGGGATGGGTCCCAAACACCTATTATCCATGTATTCAGCTGGATGCA CTGTGCTGTAGA TATGAGAAGCAGAAAGGAAGACTAAATGCTACATTACTGAAGAATGGAAGCATG hTRAV08-1 hTRAV08- 125ATGCTCCTGTTGCTCATACCAGTGCTGGGGATGATTTTTGCCCTGAGAGATGCCAGAGCCCAGTCTGTGAGCCAGATCTGAGGA GTGGAGTGACAC 1/08-3CATAACCACCACGTAATTCTCTCTGAAGCAGCCTCACTGGAGTTGGGATGCAACTATTCCTATGGTGGAACTGTTAACCCTCTG AGCTGAGTACTTAATCTCTTCTGGTATGTCCAGTACCCTGGTCAACACCTTCAGCTTCTCCTCAAGTACTTTTCAGGGGATCCACTGGTGCA CTGTGCCGTGAA TTAAAGGCATCAAGGGCTTTGAGGCTGAATTTATAAAGAGTAAATTCTCCTTTATGC hTRAV08-2 hTRAV08- 126ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGACCTGACGA ATGAGCGACGCG 2/08-4CTTGACAGCCACGTCTCTGTCTCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTATTCACCATAACCCTCAG GCTGAGTACTTCCTCTCTTCTGGTATGTGCAACACCCCAACAAAGGACTCCAGCTTCTCCTGAAGTACACATCAGCGGCCACCCTGGCCCAT TGTGTTGTGAGTTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC GA hTRAV08-3hTRAV08- 127ATGCTCCTGGAGCTTATCCCACTGCTGGGGATACATTTTGTCCTGAGAACTGCCAGAGCCCAGTCAGTGACCCAGATCTGAGGA TTGGAGTGATGC 1/08-3CCTGACATCCACATCACTGTCTCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGGGGCAACACCTAACCCTCTG TGCTGAGTACTTTATCTCTTCTGGTATGTCCAGTCCCCCGGCCAAGGCCTCCAGCTGCTCCTGAAGTACTTTTCAGGAGACACTCTGGTGCA CTGTGCTGTGGG TTCAAGGCATTAAAGGCTTTGAGGCTGAATTTAAGAGGAGTCAATCTTCCTTCATGC hTRAV08-4 hTRAV08- 128ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGACCTGACGA ATGAGCGACGCG 2/08-4CTTGGCAGCCACGTCTCTGTCTCTGAAGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTTCCACCATAACCCTCAG GCTGAGTACTTCATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTACACATCAGCGGCCACCCTGGCCCAT TGTGCTGTGAGTTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC GACACA hTRAV08-5hTRAV08-5 129ATGCTCCTGGTGCTCATCCCACTGCTGGGGATACATTTTGTCCTGAGTGAGAACTGTCAGAGCCCAGTCAGTGACCCTATGCCT TCTTAATCCTGTCCCAGCCTGACATCCGCATCACTGTCTCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGGGGCGATGTCTTTACT AGCTGAGGAGGAGTTGTGGGAAGTCAGGGACCCCAAACGGAGGGACCGGCTGAAGCCATGGCAGAAGAATGTGGATTGTGAAGATTTAATC TGTATGTCACCTTCATGGACATTTATTAGTTCCCCAAATTAATACTTTTATAATTTCTTATGCCTCTCTTTACTGCAATCTCTAAACATAAATTGTAAAGATTTCATGGACACTTATCACTTCCCCAATCAATACCCCTGTGATTT hTRAV08-6hTRAV08-6 130ATGCTCCTGCTGCTCGTCCCAGCGTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAGCTTGAGGAA AAGCGACACGGCCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGACCCTCAGT TGAGTACTTCTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTGGCCATAT TGCTGTGAGTGATTAAAGGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCA hTRAV08-7hTRAV08-7 131ATGCTCTTAGTGGTCATTCTGCTGCTTGGAATGTTCTTCACACTGAGAACCAGAACCCAGTCGGTGACCCAGCTTGAAACCATC TGCTGCTGAGTAGATGGCCACATCACTGTCTCTGAAGAAGCCCCTCTGGAACTGAAGTGCAACTATTCCTATAGTGGAGTTCCTTCTAACCCATGT CTTCTGTGCTGTGCTCTTCTGGTATGTCCAATACTCTAGCCAAAGCCTCCAGCTTCTCCTCAAAGACCTAACAGAGGCCACCCAGGTTGAGTGA GGTGACAGGAAAGGCATCAGAGGTTTTGAGGCTGAATTTAAGAAGAGCGAAACCTCCTTCTACCTGAG hTRAV09-1hTRAV09-1 132ATGAATTCTTCTCCAGGACCAGCGATTGCACTATTCTTAATGTTTGGGGGAATCAATGGAGATTCAGTGGTCCAGACTTGGAGA GAGTCAGACTCCACAGAAGGCCAAGTGCTCCCCTCTGAAGGGGATTCCCTGATTGTGAACTGCTCCTATGAAACCACACAGTACCCTAAGACTCAG GCTGTGTACTTCTTCCCTTTTTTGGTATGTCCAATATCCTGGAGAAGGTCCACAGCTCCACCTGAAAGCCATGAAGGCCAATGACAAGTTCAA GTGCTCTGAGTG GGAAGGAACAAAGGTTTTGAAGCCATGTACCGTAAAGAAACCACTTCTTTCCA hTRAV09-2 hTRAV09-2 133ATGAACTATTCTCCAGGCTTAGTATCTCTGATACTCTTACTGCTTGGAAGAACCCGTGGAAATTCAGTGACCCAGACTTGGAGA GTGTCAGACTCAATGGAAGGGCCAGTGACTCTCTCAGAAGAGGCCTTCCTGACTATAAACTGCACGTACACAGCCACAGGATACCCAAGGCTCAG GCGGTGTACTTCTTCCCTTTTCTGGTATGTCCAATATCCTGGAGAAGGTCTACAGCTCCTCCTGAAAGCCACGAAGGCTGATGACAATTCAA TGTGCTCTGAGT GGGAAGCAACAAAGGTTTTGAAGCCACATACCGTAAAGAAACCACTTCTTTCCGA hTRAV10 hTRAV10 134ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTTTATAGGGGGAATGGCAAAAACCAAGTGCTGCACATC GCTCAGCGATTCGAGCAGAGTCCTCAGTCCCTGATCATCCTGGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTGAGCCCCACAGCCTCC AGCCTCCTACATTTCAGCAACTTAAGGTGGTATAAGCAAGATACTGGGAGAGGTCCTGTTTCCCTGACAATCATGACTTTCAGTGAGCA CTGTGTGGTGAGAACACAAAGTCGAACGGAAGATATACAGCAACTCTGGATGCAGACACAAAGCAAAGCTCT CG hTRAV11hTRAV11 135ACGGAGAAGCCCTTGGGAGTTTCATTCTTGATTTCCTCCTGGCAGCTGTGCTGGGTGAATAGACTACATACACTGGTTTGGAAT CTGGGAGATTCAGAGCAGAGTCCTTCATTCCTGAATATTCAGGAGGGAATGCATGCCGTTCTTAATTGTACTTATCAGGAGAGAACAATCGCAGCC GCCACCTACTTCCTCTTCAATTTCCACTGGTTCCGGCAGGATCCGGGGAGAAGACTTGTGTCTTTGACCTTAATTCAATCAAGCCAGTCTCAT TGTGCTTTGCAAGGAGCAGGGAGACAAATATTTTAAAGAACTGCTTGGAAAAGAAAAATTTTATAGT hTRAV12-1hTRAV12-1 136ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGCCCTGCTCA CTCAGTGATTCAGAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCAGAGACT GCCACCTACCTCTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGTCCAAG TGTGTGGTGAAC AATGAAGATGGAAGGTTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTTA hTRAV12-2 hTRAV12-2 137ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGCTCTGCTCA CCCAGTGATTCAGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCAGAGACT GCCACCTACCTCTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTCCCAG TGTGCCGTGAAGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTT hTRAV12-3hTRAV12-3 138ATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACAGAAGGAGGTGCCTTGTTCA CCCAGTGATTCAGAGCAGGATCCTGGACCACTCAGTGTTCCAGAGGGAGCCATTGTTTCTCTCAACTGCACTTACAGCAACAGTGCTTCAGAGACT GCCACCTACCTCTTTCAATACTTCATGTGGTACAGACAGTATTCCAGAAAAGGCCCTGAGTTGCTGATGTACACATACTCCAGTGGTCACAG TGTGCAATGAGCAACAAAGAAGATGGAAGGTTTACAGCACAGGTCGATAAATCCAGCAAGTATATCT GCACAG hTRAV13-1hTRAV13-1 139ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGTCCCTGCAC CCTGAAGACTCGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACATCACAGAG GCTGTCTACTTCTTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAGGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAACCCAA GTGCAGCAAGTA AAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTChTRAV13-2 hTRAV13-2 140ATGGCAGGCATTCGAGCTTTATTTATGTACTTGTGGCTGCAGCTGGACTGGGTGAGCAGAGGAGAGAGTGTGGGTCTCTGCAA CCTGGAGACTCAGCTGCATCTTCCTACCCTGAGTGTCCAGGAGGGTGACAACTCTATTATCAACTGTGCTTATTCAAACAGCGCCTCATTGCAGCT GCTGTCTACTTTTAGACTACTTCATTTGGTACAAGCAAGAATCTGGAAAAGGTCCTCAATTCATTATAGACATTCGTTCAAATATGGAACTCAA GTGCAGAGAATACAAAAGGCAAGGCCAAAGAGTCACCGTTTTATTGAATAAGACAGTGAAACATCTC hTRAV14 hTRAV14141ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACTTTGTCATCT GGGACTCAGCAACAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCACCGCTTCAC TGTATTTCTGTGCAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGAAACTGG AATGAGAGAGGGCGAGCAAAATGCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAACChTRAV15 hTRAV15 142ATGTATACGTATGTAACAAACCTGCGCGTTGTGCACATGTACCCTAGAACGGGTGAACAGCCTCCATATTCTGGAGTTTTGAAT CCTGGAGATTCAGTAGAGTCCTTCATTCATTCCTGAGTATCCGGGAGGGAATGCACAACATTCTTAATTGCACTTATGAGGAGAGAAATGCTGGTC GGCACCTACTTCCGTTCTCTTAACTTCTACTGGTTCTGGCAGGGTCTGGAAAAGGACTTGTGTCTTTGACCTTAATTCAATCAAGCCATCTCAT TGTGCTTTGAGGGATGGAGGAGGGAGACAAACATTTTAAAGAAGCGCTTGGAAAAGAGAAGTTTTATAGT hTRAV16hTRAV16 143ATGAAGCCCACCCTCATCTCAGTGCTTGTGATAATATTTATACTCAGAGGAACAAGAGCCCAGAGAGTGACTCACCTGAAGAA GGAAGACTCAGCGCCCGAGAAGCTCCTCTCTGTCTTTAAAGGGGCCCCAGTGGAGCTGAAGTGCAACTATTCCTATTCTGGGAGTCCACCATTTGC CATGTATTACTGTGAACTCTTCTGGTATGTCCAGTACTCCAGACAACGCCTCCAGTTACTCTTGAGACACATCTCTAGAGAGAGCATTCAAGA TGCTCTAAGTGG CAAAGGCTTCACTGCTGACCTTAACAAAGGCGAGACATCTTTCCAhTRAV17 hTRAV17 144ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTGGCTAGGGTGAACAGTCAACAGGGAGAATCCTTGTTG GCAGCAGACACTGAGGATCCTCAGGCCTTGAGCATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGTATAAAATCACGGCT GCTTCTTACTTCTCAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGATCCCGG GTGCTACGGACG GAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGThTRAV18 hTRAV18 145ATGCTGTCTGCTTCCTGCTCAGGACTTGTGATCTTGTTGATATTCAGAAGGACCAGTGGAGACTCGGTTACCCAGACCTGGAGA GCTGTCGGACTCACAGAAGGCCCAGTTACCCTCCCTGAGAGGGCAGCTCTGACATTAAACTGCACTTATCAGTCCAGCTATTCAACTAGCCCTCGG TGCCGTGTACTATTTCTATTCTGGTATGTCCAGTATCTAAACAAAGAGCCTGAGCTCCTCCTGAAAAGTTCAGAAAACCAGGAGACGTGCA CTGCGCTCTGAG GACAGCAGAGGTTTTCAGGCCAGTCCTATCAAGAGTGACAGTTCCTTCC AhTRAV19 hTRAV19 146ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCACCATCAC GGACTCAGCAGTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACAGCCTCACA ATACTTCTGTGCTTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATAGTCGT CTGAGTGAGGCGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTThTRAV20 hTRAV20 147ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTGTTTCTGCAC AACCTGAAGACTACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTTAACTGCAGTTACACAGTCAGCGGATCACAGCC CAGCCACTTATCTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGGCCTA TCTGTGCTGTGC GGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGCAGG hTRAV21 hTRAV21 148ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCACTTTATACA TGGTGACTCAGCGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTATTGCAGCTT CACCTACCTCTGCAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGACTCAGCC TGCTGTGAGG GCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTAhTRAV22 hTRAV22 149ATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTTTGCTGTGTGAGAGGAATACAAGTGGAGTACATTTC AGACTCAGGCGTGCAGAGTCCTCCAGACCTGATTCTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCTGTGAACTCTTCCCA TTATTTCTGTGCTCAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATCAACCTGTTTTACATTCCCTCAGGGACAAAACAGACCAC GTGGAGC GAATGGAAGATTAAGCGCCACGACTGTCGCTACGGAACGCTACAGCTTATThTRAV23 hTRAV23 150ATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTATGCTGGGTGAGTGGCCAACAGAAGGAGCATTGCATA CTGGAGACTCAGAAAAGTGACCAGCAGCAGGTGAAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATTATAAATCATGGATT CCACCTACTTCTCTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTACCAACAATTCCCTGGGAAAGGCCCTGCATTATTCCCAGC GTGCAGCAAGCAGATAGCCATACGTCCAGATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAAAGTGCCAAGCAGTTCT hTRAV24 hTRAV24 151ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAACGCTATTTGT AGCCTGAAGACTGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCACATCAAAG CAGCCACATACCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGATCCC TCTGTGCCTTTAGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACA hTRAV25hTRAV25 152ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCAGCTCCCT CCCAGACTACAGCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATAGCACATCAC ATGTAGGAACCTCAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAAGCCA ACTTCTGTGCAG GCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAA GGhTRAV26-1 hTRAV26-1 153ATGAGGCTGGTGGCAAGAGTAACTGTGTTTCTGACCTTTGGAACTATAATTGATGCTAAGACCACCCAGCCCCCCTTGATCCTG GAGACACTGCTGTCCATGGATTGCGCTGAAGGAAGAGCTGCAAACCTGCCTTGTAATCACTCTACCATCAGTGGAAATGAGTATGTGCCCCACGCT TGTACTATTGCATATTGGTATCGACAGATTCACTCCCAGGGGCCACAGTATATCATTCATGGTCTAAAAAACAATGAAACCAATGAACGCTGA TCGTCAGAGTCG AATGGCCTCTCTGATCATCACAGAAGACAGAAAGTCCAGCACChTRAV26-2 hTRAV26-2 154ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATGGGTGATGCTAAGACCACACAGCCAAATTTGATCCTG GAGATGCTGCTGTCAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACACCGTGCT TGTACTACTGCACATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAGCAATGTGAACAACAGAACCTTGA TCCTGAGAGAC ATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACC hTRAV27hTRAV27 155ATGGTCCTGAAATTCTCCGTGTCCATTCTTTGGATTCAGTTGGCATGGGTGAGCACCCAGCTGCTGGAGCAGAGCGTTCTCTCC CAGCCTGGTGATCCTCAGTTTCTAAGCATCCAAGAGGGAGAAAATCTCACTGTGTACTGCAACTCCTCAAGTGTTTTTTCCAGCTTACACATCACTG ACAGGCCTCTACAATGGTACAGACAGGAGCCTGGGGAAGGTCCTGTCCTCCTGGTGACAGTAGTTACGGGTGGAGAAGTGAAGAAGCAGCC CTCTGTGCAGGA CTGAAGAGACTAACCTTTCAGTTTGGTGATGCAAGAAAGGACA G hTRAV28hTRAV28 156ATGAAGGCATTAATAGGAATCTTGCTGGGCTTCCTGTGGATACAGATTTGCTCGCAAATGAAAGTGGAGCAGAGGCCACCTAT GCCTGAGGACTCTCCTCAGGTCCTGATCCTCCAAGAGGGAAGAAATTCATTCCTGGTGTGCAGTTGTTCTATTTACATGATCCGTGTGACATCAGAT AGCTATTTACTTCCAGTGGTTTCATCAAAAGCCTGGAGGACCCCTCATGTCCTTATTTAACATTAATTCAGGAATACAGCAAAAAAGATCCCA TGTGCTGTGGGG AGACTAAAATCCGCAGTCAAAGCTGAGGAACTTTATG A hTRAV29hTRAV29 157ATGGCCATGCTCCTGGGGGCATCAGTGCTGATTCTGTGGCTTCAGCCAGACTGGGTAAACAGTCAACAGAAGAATCTCTGCAC GCCTGGAGACTCTGATGACCAGCAAGTTAAGCAAAATTCACCATCCCTGAGCGTCCAGGAAGGAAGAATTTCTATTCTGAACTGTGATTGTGCCC TGCAGTGTACTTACTATACTAACAGCATGTTTGATTATTTCCTATGGTACAAAAAATACCCTGCTGAAGGTCCTACATTCCTGATATCTCCCA CTGTGCAGCAAGTATAAGTTCCATTAAGGATAAAAATGAAGATGGAAGATTCACTGTCTTCTTAAACAAAAGTGCCAAGCACCTCCG hTRAV30 hTRAV30 158ATGGAGACTCTCCTGAAAGTGCTTTCAGGCACCTTGTTGTGGCAGTTGACCTGGGTGAGAAGCCAACAACCAGTGCCCTGTACC CAGTTACTCAGGCAGAGTCCTCAAGCCGTGATCCTCCGAGAAGGGGAAGATGCTGTCATCAACTGCAGTTCCTCCAAGGCTTTATATTTACGGCCT AACCTACTTCTGTCTGTACACTGGTACAGGCAGAAGCATGGTGAAGCACCCGTCTTCCTGATGATATTACTGAAGGGTGGAGAACACCCAGCT CGGCACAGAGAGAAGGGTCATGAAAAAATATCTGCTTCATTTAATGAAAAAAAGCAGCAAAGCT hTRAV31 hTRAV31159ATGACTGTTGGCAGCATATTACGGGCACTCATGGCCTCTGCCTTCCTTGCATGTCACAGAGGGTCATTCAATCCCCTTATCATA GAAGACCTGCAAAACCAGCAATATCTACGCAGGAGGGTGAGACCGTGAAACTGGACTGTGCATACAAAACTAATATTGTATATTACTCATCATCA CATATTTCTGTTGATATTGTATTGGTACAAAAGGTCTCCCAATGGGAAGATTATTTTCCTCATTTATCAGCAAACAGATGCAGAAACCCAGCCA TCTCAAAGAGCCAATGCGACACAGGGTCAATATTCTGTGAGCTTCCAGAAAACAACTAAAACTATTCAG hTRAV32hTRAV32 160ATGGCAAGAAGAATGGAAAAGTCCCTGGGAGCTTTATTCAAATTCAGCTGAAGCTGGCCAAGAAAAGGATGTGATCCCTGCAT CCAGGAGACTCATACAGAGTTATTCAAATCTAAATGTCTAGGAGAGAGAAATGGCCGTTATTAATGACAGTTATACAGATGGAGCTTATTACAGCC TTCCTGTACTTCTTGAATTATTTCTGTTGGTACAAGAAGAAAACGGGGAAGGCCCTAATATCTTAATGGAGATTCATTCAAATGTGGAACCCAA GTGCAGTGAGAATAGAAAACAGGACAGAAGGCTCACTGTACTGTTGAATAAAAATGCTAAACATGTC CACA hTRAV33hTRAV33 161ATGCTCTGCCCTGGCCTGCTGTGGGCATTCGTGGTCCCCTTTGGCTTCAGATCCAGCATGGCTCAGAAAGTAACCACCTCACCA TGACTCAGCCAACAAGTTCAGACCACAGTAACTAGGCAGAAAGGAGTAGCTGTGACCTTGGACTGCATGTTTGAAACCAGATAGAATCAATTCCT GTACTTCTGTGCTTTCGTACACTTTATACTGGTACAAGCAACAAGCAACCTCCCAGTGAAGAGATGGTTTTCCTTATTCATCAGGGTTTAAAAC CTCAGGAATCCATTCTAAGTCAAATGCAAAGCCTGTGAACTTTGAAAAAAAGAAAAAGTTCATCA hTRAV34 hTRAV34162ATGGAGACTGTTCTGCAAGTACTCCTAGGGATATTGGGGTTCCAAGCAGCCTGGGTCAGTAGCCAAGAACTGGATCCCTGCAT CCCAGCCATGCAGCAGAGTCCTCAGTCCTTGATCGTCCAAGAGGGAAAGAATCTCACCATAAACTGCACGTCATCAAAGACGTTATATCACAGCC GGCATCTACCTCATGGCTTATACTGGTATAAGCAAAAGTATGGTGAAGGTCTTATCTTCTTGATGATGCTACAGAAAGGTGGGGAATCCCAG TGTGGAGCAGACGAGAAAAGTCATGAAAAGATAACTGCCAAGTTGGATGAGAAAAAGCAGCAAAGT A hTRAV35 hTRAV35163ATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGTCTTCCTGAA ACCTAGTGATGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGTATCTCAGC AGGCATCTACTTCTATGGTACAAGCAGGAACCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAATCCAT CTGTGCTGGGCA AATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAG GhTRAV36 hTRAV36 164ATGATGAAGTGTCCACAGGCTTTACTAGCTATCTTTTGGCTTCTACTGAGCTGGGTGAGCAGTGAAGACAAGGTGTCCTGAACA ACCGGAGACTCGGTACAAAGCCCTCTATCTCTGGTTGTCCACGAGGGAGACACCGTAACTCTCAATTGCAGTTATGAAGTGACTAACTCACAGCCA GCCATCTACCTCTTTCGAAGCCTACTATGGTACAAGCAGGAAAAGAAAGCTCCCACATTTCTATTTATGCTAACTTCAAGTGGAATTCCCAG TGTGCTGTGGAGGAAAAGAAGTCAGGAAGACTAAGTAGCATATTAGATAAGAAAGAACTTTCCAGCA G hTRAV37hTRAV37 165ATGGAAACTCCACTGAGCACTCTGCTGCTGCTCCTCTGTGTGCAGCTGACCTGGTCAAATGGACAACTGCCAGTGTCCCTGCAC CTCCATGACTCAGAACAGAATGCTCCTTCCCTGAAAGTCAAGGAAGGTGACAGCGTCACACTGAACTGCAGTTACAGAGACAGCCCATACAGGAT ACCACATTCTTCTTTCAGATTTCTTCAGTGGTTCAGGCAGGATCCTGAGGAAGGCCTCATTTCCCTGATACAAATGCTATCAACTGTGTCCCAG GCGCAGCAAGCAAGAGAGAAGATCAGTGGAAGATTCACAGCCAGGCTTAAAAAAGGAGACCAGCACATT hTRAV38-1hTRAV38 166ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAAGATCTC GGGACACTGCGACAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAAAGACTCACA TGTATTTCTGTGCTAATTATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCTGG TTTCATGAAGCAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCThTRAV38-2 hTRAV38 167ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGCATGGCTCAGACAGTCACTCCAAGATCTC GGGATGCCGCGAAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAGAGTAGACTCACA TGTATTTCTGTGCGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGGCTGG TTATAGGAGCGCAACAGAATGCAACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCThTRAV39 hTRAV39 168ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTAAGTGGAGAGCTGAAAGTGGAACAAAACCGTCTCAG CAGCTGCCGTGCCCCTCTGTTCCTGAGCATGCAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGACAGACTCACCCTCCA ATGACCTCTCTGGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCACATCA CCACCTACTTCT GGAGGGACGATTAATGGCCTCACTTGATACCAAAGC GTGCCGTGGACAhTRAV40 hTRAV40 169ATGAACTCCTCTCTGGACTTTCTAATTCTGATCTTAATGTTTGGAGGAACCAGCAGCAATTCAGTCAAGCAGACGCCATTGTGA TATCAGACTCAGGGCCAAATAACCGTCTCGGAGGGAGCATCTGTGACTATGAACTGCACATACACATCCACGGGGTACCCTACCCTTAATATTCAG CCGTGTACTACTTTCTGGTATGTGGAATACCCCAGCAAACCTCTGCAGCTTCTTCAGAGAGAGACAATGGAAAACAGCAAAAACTTTCCAGG GTCTTCTGGGAG CGGAGGCGGAAATATTAAAGACAAAAACTCCC A hTRAV41 hTRAV10170ATGGTGAAGATCCGGCAATTTTTGTTGGCTATTTTGTGGCTTCAGCTAAGCTGTGTAAGTGCCGCCAAAAATGAACTGCACATC TCCCAGAGACTCGTGGAGCAGAGTCCTCAGAACCTGACTGCCCAGGAAGGAGAATTTATCACAATCAACTGCAGTTACTCGGTAGGACAGCCTCC TGCCGTCTACATAATAAGTGCCTTACACTGGCTGCAACAGCATCCAGGAGGAGGCATTGTTTCCTTGTTTATGCTGAGCTCAGGGAACA CTGTGCTGTCAG GAAGAAGCATGGAAGATTAATTGCCACAATAAACATACAGGAAAAGCACAGCTCCA

TABLE 16 TCR βchain V segments and binding sites for primers presented in Table 4 and Table 10. The sequence for each V segmentspresented in this table consists of three parts (listed from 5′to 3′): Sequence upstream of primer binding site, sequence ofthe primer binding site and sequence downstream of the primer binding site.V SEQ Primer hTRbV sequence segment Primer ID binding site downstream ofname name NO hTRBV sequence upstream of primer binding site within hTRbVprimer binding site hTRBV01 hTRBV01 171ATGGGCTGAAGTCTCCACTGTGGTGTGGTCCATTGTCTCAGGCTCCATGGATACTGGAATTACCCAGACACCAAAGTGGTCGCA CTCAGCTGCGTAATACCTGGTCACAGCAATGGGGAGTAAAAGGACAATGAAACGTGAGCATCTGGGACATGATTCTATGTATTGGTCTGCAGCAA TCTCTGCACCAGACAGACAGAAAGCTAAGAAATCCCTGGAGTTCATGTTTTACTACAACTGTAAGGAATTCATTGAAAACAAGACTGAAGA CAGCCAAGA GTGCCAAATCACTTCACACCTGAATGCCCTGACAGCTCTCGCTTATACCTTCAThTRBV02 hTRBV02 172ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAGGATCCGGTC CTCAGCCATGTAACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACCACAAAGCT CTTCTGTGCCAGTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGGGAGGA CAGTGAAGCAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAAhTRBV03-1 hTRBV03-1 173ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCAGGTCCCTTGGACACAGCTGTTTCCCAGACATCAATTC CTCTGCTGTGTATCTCCAAAATACCTGGTCACACAGATGGGAAACGACAAGTCCATTAAATGTGAACAAAATCTGGGCCATGATACTCCTGGAGCT TTCTGTGCCAGCATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGATAATGTTTAGCTACAATAATAAGGAGCTCATTATATGGTGA AGCCAAGAAATGAAACAGTTCCAAATCGCTTCTCACCTAAATCTCCAGACAAAGCTCACTTAAATCTTCA hTRBV03-2hTRBV03-1 174ATGGGCTGCAGGCTCCTCTGCTATGTGGCCCTCTGCCTCCTGCAAGCAGGATCCACTGGACACAGCCGTTTCCCACATCAATTC CTCTGCTGTGTATGACTCCAAAATACCTGGTCACACAGATGGGAAAAAAGGAGTCTCTTAAATGAGAACAAAATCTGGGCCATAATGCCTGGAGCT TTCTGTGCCAGCCTATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGACAATGTTTATCTACAGTAACAAGGAGCCAATTTTGGTGA AGCCAAGATAAATGAAACAGTTCCAAATCGCTTCTCACCTGACTCTCCAGACAAAGCTCATTTAAATCTTCAhTRBV04-1 hTRBV04-1 175ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGACACTGAAGTTACCCAGTTCACCTAC AAGACTCAGCCCACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCACAGGGCACGCCCTGC TGTATCTCTGCGTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTATAGCCAG CCAGCAGCCAAGAAATGAAAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACC A hTRBV04-2hTRBV04-2 176ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGTCCCCATGGAAACGGGAGTTACGCAGTTCACCTAC AAGACTCGGCCCACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGGCATAACGCACACCCTGC TGTATCTCTGTGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGTCTACAACTTTAAAGAACAGACTGAAGCCAG CAGCAGCCAAGAAAACAACAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATTCC hTRBV04-3hTRBV04-2 177ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGGTGAGTTGGTCCCCATGGAAACGGGATTCACCTAC AAGACTCGGCCCGTTACGCAGACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGACACCCTGC TGTATCTCTGCGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGTCTACAGTCTTGAAGAAGCCAG CCAGCAGCCAAGACGGGTTGAAAACAACAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATTCC AhTRBV05-1 hTRBV05-1 178ATGGGCTCCAGGCTGCTCTGTTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTAAAGGCTGGAGTCACTCAAGAATGTGAG GGGACTCGGCCCACTCCAAGATATCTGATCAAAACGAGAGGACAGCAAGTGACACTGAGCTGCTCCCCTATCTCTGGGCATAGGAGCACCTTGGA TTTATCTTTGCGCTGTATCCTGGTACCAACAGACCCCAGGACAGGGCCTTCAGTTCCTCTTTGAATACTTCAGTGAGACACAGAGAAAGCTGG CAGCAGCTTGGCAAAGGAAACTTCCCTGGTCGATTCTCAGGGCGCCAGTTCTCTAACTCTCGCTCTGAGAT hTRBV05-2hTRBV05-2 179ATGGGCTCCGGACTCCTCTGCTGGACGCTGCTTTGTTTCCTGGGAGCAGGCCCAGTGGAGGCTGGAATCACCCAATACTGAGTC GGACTCAGCCCTGCTCCAAGACACCTGATCAAAACAAGAGACCAGCAAGTGACACTGAGATGCTCCCCTGCCTCTGGGCATAACTGAAACACGGA GTATCTCTGTGCTGTGTCCTGGTACCTACGAACTCCAAGTCAGCCCCTCTAGTTATTGTTACAATATTGTAATAGGTTACAAAGAGCGCTAGG CAGCAACTTG AAAAGGAAACTTGCCTAATTGATTCTCAGCTCACCACGTCCATAACTAThTRBV05-3 hTRBV05-3 180ATGGGCCCCGGGCTCCTCTGCTGGGAACTGCTTTATCTCCTGGGAGCAGGCCCAGTGGAGGCTGGAGTCACCCAAGCTCTGAGA TGGAGCTGGGGGAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCACAGCAGTGAATGTGA ACTCGGCCCTGTTGTGTCCTGGTACCAACAGGCCCCGGGTCAGGGGCCCCAGTTTATCTTTGAATATGCTAATGAGTTAAGGAGATCGTGCCT ATCTCTGTGCCA AGAAGGAAACTTCCCTAATCGATTCTCAGGGCGCCAGTTCCATGACTGTTGAAGCTTGG hTRBV05-4 hTRBV05-4 181ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCTCAGTGGAGACTGGAGTCACCCAACTGAGCTGA GGAGCTGGACGAAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTTCTCAGTCTGGGCACAACACATGTGAACG CTCGGCCCTGTATGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATAGGGAGGAAGAGAATGGCCTT TCTCTGTGCCAG CAGAGGAAACTTCCCTCCTAGATTCTCAGGTCTCCAGTTCCCTAATTATAGCTCAGCTTGG hTRBV05-5 hTRBV05-4 182ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTGGACGCTGGAGTCACCCAACTGAGCTGA GTTGCTGGGGGAAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCACAAGAGATGTGAACG CTCGGCCCTGTATGTGTCCTGGTACCAACAGGTCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGAAAGAAGAGAGAGCCTT TCTCTGTGCCAG GAAGAGGAAACTTCCCTGATCGATTCTCAGCTCGCCAGTTCCCTAACTATAGCTCAGCTTGG hTRBV05-6 hTRBV05-4 183ATGGGCCCCGGGCTCCTCTGCTGGGCACTGCTTTGTCTCCTGGGAGCAGGCTTAGTGGACGCTGGAGTCACCCAACTGAGCTGA GTTGCTGGGGGAAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTAAGTCTGGGCATGACACATGTGAACG CTCGGCCCTCTATGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGGAGGAAGAGAGACCCTT TCTCTGTGCCAG AGAGAGGCAACTTCCCTGATCGATTCTCAGGTCACCAGTTCCCTAACTATAGCTCAGCTTGG hTRBV05-7 hTRBV05-4 184ATGGGCCCCGGGCTCCTCTGCTGGGTGCTGCTTTGTCCCCTAGGAGAAGGCCCAGTGGACGCTGGAGTCACCCAACTGAGCTGA GTTGCTAGGGGAAGTCCCACACACCTGATCAAAACGAGAGGACAGCACGTGACTCTGAGATGCTCTCCTATCTCTGGGCACACCAGATGTGAACG CTCGGCCCTCTATGTGTCCTCGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGAAAGAAGAGAGAGCCTT TCTCTGTGCCAG GAAGAGGAAACTTCCCTGATCAATTCTCAGGTCACCAGTTCCCTAACTATAGCTCAGCTTGG hTRBV05-8 hTRBV05-4 185ATGGGACCCAGGCTCCTCTTCTGGGCACTGCTTTGTCTCCTCGGAACAGGCCCAGTGGAGGCTGGAGTCACACAACTGAGCTGA GGAGCTGGAGGAAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGCGACTCTGAGATGCTCTCCTATCTCTGGGCACACCAGATGTGAACG CTCGGCCCTGTATGTGTACTGGTACCAACAGGCCCTGGGTCTGGGCCTCCAGTTCCTCCTTTGGTATGACGAGGGTGAAGAGAGAAACCTT TCTCTGTGCCAG CAGAGGAAACTTCCCTCCTAGATTTTCAGGTCGCCAGTTCCCTAATTATAGCTCAGCTTGG hTRBV06-1 hTRBV06-1 186ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAAGTCCAGTGAATGCTGGTGTCACTCAGGAGTTCTCG CGGCTGCTCCCTACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATAACTCCTCAGGCTG CCCAGACATCTGCATGTACTGGTATCGACAAGACCCAGGCATGGGACTGAGGCTGATTTATTACTCAGCTTCTGAGGGTACCACTGAGAGT TGTACTTCTGTGC CAAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATTAAACAAACGGCAGCAGTGAAGC hTRBV06-2 hTRBV06-2 187ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGCTGGGGTTG CCTCCCAAACATACCCCAAAATTCCGGGTCCTGAAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATAGAGTCGGCT CTGTGTACTTCTGCATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGAGGGTACAACTGGCTC TGCCAGCAGTTACCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG CTChTRBV06-3 hTRBV06-2 188ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGCTGGGGTTG CCTCCCAAACATACCCCAAAATTCCGGGTCCTGAAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATAGAGTCGGCT CTGTGTACTTCTGCATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGAGGGTACAACTGGCTC TGCCAGCAGTTACCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG CTChTRBV06-4 hTRBV06-4 189ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAGCCCCTCACG TACCCTCTCAGAGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCTTGGCGTCT CATCTGTGTACTTCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGGCTG CTGTGCCAGCAGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTC TGACTC hTRBV06-5hTRBV06-5 190ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGTCCCGCTCA TGCTCCCTCCCAACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATAGGCTGCTGT GACATCTGTGTACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACGGC CTTCTGTGCCAG CCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTCAGTTACTC hTRBV06-6 hTRBV06-6 191ATGAGCATCAGCCTCCTGTGCTGTGCAGCCTTTCCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGGATTTCCCG TGGCTGCTCCCTACCCCAAAATTCCGCATCCTGAAGATAGGACAGAGCATGACACTGCAGTGTACCCAGGATATGAACCATAACTACTCAGGCTG CCCAGACATCTGCATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAAGCTGATTTATTATTCAGTTGGTGCTGGTATCACTGAGAGT TGTACTTCTGTGC TAAAGGAGAAGTCCCGAATGGCTACAACGTCTCCAGATCAACCACAGAGCAGCAGTTACTC hTRBV06-7 hTRBV06-7 192ATGAGCCTCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAATGAATGCTGGTGTCACTCAGATCCCCCTCA GCTCCCTCTCAGCCCCAAAATTCCACGTCCTGAAGACAGGACAGAGCATGACTCTGCTGTGTGCCCAGGATATGAACCATGAATACAGCTGGAGT ACTTCTGTTTACTATGTATCGGTATCGACAAGACCCAGGCAAGGGGCTGAGGCTGATTTACTACTCAGTTGCTGCTGCTCTCACTGACCAGCT TCTGTGCCAGCA AAAGGAGAAGTTCCCAATGGCTACAATGTCTCCAGATCAAACACAGAGGATTGTTACTC hTRBV06-8 hTRBV06-8 193ATGAGCCTCGGGCTCCTGTGCTGTGCGGCCTTTTCTCTCCTGTGGGCAGGTCCCGTGAATGCTGGTGTCACTCAGATCCCACTCA TGCTCCCTCCCACCCCAAAATTCCACATCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGGATACGGCTGGTGT GACATCTGTGTAATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGACTGATTTACTACTCAGCTGCTGCTGGTACTACTGACCGGC CTTGTGTGCCAG AAAGAAGTCCCCAATGGCTACAATGTCTCTAGATTAAACACAGAGGATTCAGTTACTC hTRBV06-9 hTRBV06-6 194ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGAGGGTCCAGTGAATGCTGGTGTCACTCAGGATTTCCCG CAGCTGCTCCCTACCCCAAAATTCCACATCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGGATACTCAGGCTG CCCAGACATCTGCTTGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCGCATTCATTACTCAGTTGCTGCTGGTATCACTGAGAGT TATACTTCTGTGC CAAAGGAGAAGTCCCCGATGGCTACAATGTATCCAGATCAAACACAGAGCAGCAGTTATTC hTRBV07-1 hTRBV07-1 195ATGGGCACAAGGCTCCTCTGCTGGGCAGCCATATGTCTCCTGGGGGCAGATCACACAGGTGCTGGAGTCTCCCACTCTGAAGT GCAGGGGGACTTGTCCCTGAGACACAAGGTAGCAAAGAAGGGAAAGGATGTAGCTCTCAGATATGATCCAATTTCAGGTCATAATGTCCAGCGCA GGCTGTGTATCTCCCTTTATTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTTCCAATTTACTTCCAAGGCAAGGATGCAGCAGCACA CTGTGCCAGCAGACAAATCGGGGCTTCCCCGTGATCGGTTCTCTGCACAGAGGTCTGAGGGATCCATCTCCA CTCAGChTRBV07-2 hTRBV07-2 196ATGGGCACCAGGCTCCTCTTCTGGGTGGCCTTCTGTCTCCTGGGGGCAGATCACACAGGAGCTGGAGTCTCCCAGGATCCAGCG GACTCGGCCGTGTCCCCCAGTAACAAGGTCACAGAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGCCACACAGCA TATCTCTGTGCCCCTTTACTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTTTTAATTTACTTCCAAGGCAACAGTGCACCAGAGGAG AGCAGCTTAGCCAAATCAGGGCTGCCCAGTGATCGCTTCTCTGCAGAGAGGACTGGGGGATCCGTCTCCACTCTGAChTRBV07-3 hTRBV07-5 197ATGGGCACCAGGCTCCTCTGCTGGGCAGCCCTGTGCCTCCTGGGGGCAGATCACACAGGTGCTGGAGTCTCCCAGACTCTGAAG GCGGGGGGACTCACCCCCAGTAACAAGGTCACAGAGAAGGGAAAATATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGCATCCAGCGC AGCCGTGTATCTCCTTTACTGGTACCGACAAAGCCTGGGGCAGGGCCCAGAGTTTCTAATTTACTTCCAAGGCACGGGTGCGGCAGACAGA CTGTGCCAGCAGATGACTCAGGGCTGCCCAACGATCGGTTCTTTGCAGTCAGGCCTGAGGGATCCGTCTCT CTTAAChTRBV07-4 hTRBV07-5 198ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGACTCTGAAG GCAGGGGGACTCTCCCCAAGGTACAAAGTCGCAAAGAGGGGACGGGATGTAGCTCTCAGGTGTGATTCAATTTCGGGTCATGTAACATCCAGCGC AGCTGTGTATCTCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCTCAGAGGTTCTGACTTACTCCCAGAGTGATGCTCAACGAGAACAGA CTGTGCCAGCAGCAAATCAGGGCGGCCCAGTGGTCGGTTCTCTGCAGAGAGGCCTGAGAGATCCGTCTCC CTTAGChTRBV07-5 hTRBV07-5 199ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGAGATCCAGC GCGACTCGGCTGTCCCCAAGGTACGAAGTCACACAGAGGGGACAGGATGTAGCTCCCAGGTGTGATCCAATTTCGGGTCAGGTAACGCACAGAGC TGTATCTCTGTGCCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCAAGAGTTTCTGACTTCCTTCCAGGATGAAACTCAACAAGAAAGG CAGAAGCTTAGTAAATCAGGGCTGCTCAGTGATCAATTCTCCACAGAGAGGTCTGAGGATCTTTCTCCACCTGAhTRBV07-6 hTRBV07-6 200ATGGGCACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGCAGCGCACA CGGCCATGTATCTCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCGAGCAGCGG GCTGTGCCAGCACTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCCCAACAAGACGACT GCTTAGCAAATCAGGGCTGCCCAATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATChTRBV07-7 hTRBV07-6 201ATGGGTACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGCAGCGCACA CAGCCATGTATCTCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAACTCTCAGGTGTGATCCAATTTCGAGTCATGCAACGAGCAGCGG GCTGTGCCAGCACCTTTATTGGTATCAACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCTCAACCAGAGACT GCTTAGCCAAATCAGGGCTGCCCAGTGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATThTRBV07-8 hTRBV07-2 202ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGGATCCAGCG GACTCCGCCGTGTCCCCTAGGTACAAAGTCGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCACACAGCA TATCTCTGTGCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGACGGAG AGCAGCTTAGCAAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTGAAhTRBV07-9 hTRBV07-9  203ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGAGATCCAG GGGACTCGGCCAAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCGCACAGAG TGTATCTCTGTGCCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGACAGG CAGCAGCTTAGCAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGhTRBV08-1 hTRBV08-1 204GAGGCAGGGATCAGCCAGATACCAAGATATCACAGACACACAGGGAAAAAGATCATCCTGAAATATGCTCAGACCCTCAACC GCACCAGCCAGATTAGGAACCATTATTCAGTGTTCTGTTATCAATAAGACCAAGAATAGGGGCTGAGGCTGATCCATTATTCAGGTACTGGAGTCT CCTCTGTACCTCTGTATTGGCAGCATGACCAAAGGCGGTGCCAAGGAAGGGTACAATGTCTCTGGAAACAAGCTCAAGCATTTTACTA GTGGCAGTGCAT C hTRBV08-2 hTRBV08-2 205ATGAACCCCAAACTCTTCTGTGTGACCCTTTGTCTCCTGGGAGCAGGCTCTATTGATGCTGGGATCACCCAGATGTCCCCAATC GCACCAGCCAGACCAAGATATCACATTGTACAGAAGAAAGAGATGATCCTGGAATGTGCTCAGGTTAGGAACAGTGTTCTGATATCCTGGCATCC CCTATCTGTACCGACAGGACCCAAGACGGGGGCTGAAGCTTATCCACTATTCAGGCAGTGGTCACAGCAGGACCAAAGTTGATGTCACCA ACTGTGGCAGCA ACAGAGGGGTACTGTGTTTCTTGAAACAAGCTTGAGCATT CATC hTRBV09hTRBV09 206ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAACTAAACCTG GGGGGACTCAGCACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCAGCTCTCTG TTTGTATTTCTGTTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGGAGCT GCCAGCAGCGTACAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAA G hTRBV10-1hTRBV10-1 207ATGGGCACGAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCAGGACACAGGGATGCTGAAATCACCCAGCCCTCACTC CTCCTCCCAGACAGCCCAAGACACAAGATCACAGAGACAGGAAGGCAGGTGACCTTGGCGTGTCACCAGACTTGGAACCACAACATGGAGTCTG ATCTGTATATTTCATATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTGATCCATTACTCATATGGTGTTCAAGACACTACTGC TGCGCCAGCAGTACAAAGGAGAAGTCTCAGATGGCTACAGTGTCTCTAGATCAAACACAGAGGACCTCC GAGTChTRBV10-2 hTRBV10-2 208ATGGGCACCAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCAGGACACAGGGATGCTGGAATCACCCAGCCCTCACTC CCGCTCCCAGACAGCCCAAGATACAAGATCACAGAGACAGGAAGGCAGGTGACCTTGATGTGTCACCAGACTTGGAGCCACAGCTATGGAGTCAG ATCTGTGTATTTCTATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTGATCTATTACTCAGCAGCTGCTGATATTACAGACTAC TGCGCCAGCAGTTAAAGGAGAAGTCCCCGATGGCTATGTTGTCTCCAGATCCAAGACAGAGAATTTCC GAGTC hTRBV10-3hTRBV10-3 209ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAGTCCTCACTC CAGCTCCCAGACAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCTTGGAGTCCG ATCTGTGTACTTCATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGCTAC TGTGCCATCAGTACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCC GAGTChTRBV11-1 hTRBV11-1 210ATGAGCACCAGGCTTCTCTGCTGGATGGCCCTCTGTCTCCTGGGGGCAGAACTCTCAGAAGCTGAAGTTGCCCAGCCACTCTCA GAGCTTGGGGACTCCCCCAGATATAAGATTACAGAGAAAAGCCAGGCTGTGGCTTTTTGGTGTGATCCTATTTCTGGCCATGCTACCAGATCCAGC TCGGCCATGTATCTTTACTGGTACCGGCAGATCCTGGGACAGGGCCCGGAGCTTCTGGTTCAATTTCAGGATGAGAGTGTAGTAGATCTGCA CTCTGTGCCAGCGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT AGCTTAGChTRBV11-2 hTRBV11-1 211ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCAGAACTCACAGAAGCTGGAGTTGCCCACCACTCTCA AAGCTTGAGGACGTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCATGCTACAGATCCAGC TCGGCCGTGTATCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTTCTGATTCAGTTTCAGAATAACGGTGTAGTGGACTGCA CTCTGTGCCAGCTGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT AGCTTAGAhTRBV11-3 hTRBV11-1 212ATGGGTACCAGGCTCCTCTGCTGGGTGGCCTTCTGTCTCCTGGTGGAAGAACTCATAGAAGCTGGAGTGGTTCAGCCACTCTCA GAGCTTGGGGACTCTCCCAGATATAAGATTATAGAGAAAAAACAGCCTGTGGCTTTTTGGTGCAATCCTATTTCTGGCCACAATACCAGATCCAGC TCGGCCGTGTATCTTTACTGGTACCTGCAGAACTTGGGACAGGGCCCGGAGCTTCTGATTCGATATGAGAATGAGGAAGCAGTAGACTGCA CTCTGTGCCAGCCGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT AGCTTAGAhTRBV12-1 hTRBV12-1 213ATGGGCTCTTGGACCCTCTGTGTGTCCCTTTATATCCTGGTAGCGACACACACAGATGCTGGTGTTATCCAGTCACGAGGATCCA GGGACTTGGGCCCCAGGCACAAAGTGACAGAGATGGGACAATCAGTAACTCTGAGATGCGAACCAATTTCAGGCCACAATGATCTTGCCCATGGA TATATTTCTGTGCCTCTGGTACAGACAGACCTTTGTGCAGGGACTGGAATTGCTGAATTACTTCTGCAGCTGGACCCTCGTAGATGACACCCA CAGCAGCTTTGCTCAGGAGTGTCCAAGGATTGATTCTCAGCACAGATGCCTGATGTATCATTCTCCACTCT hTRBV12-2hTRBV12-2 214ATGGACTCCTGGACCCTCTGTGTGTCCCTTTGTATCCTGGTAGCGACATGCACAGATGCTGGCATTATCCAGTCACCTGAAGATC AGGGGGACTCGGCCAAGCATGAGGTGACAGAAATGGGACAAACAGTGACTCTGAGATGTGAGCCAATTTTTGGCCACAATTTCCTTTCAGCCTGCA CCGTGTATGTCTTCTGGTACAGAGATACCTTCGTGCAGGGACTGGAATTGCTGAGTTACTTCCGGAGCTGATCTATTATAGATAATGGAGC GTGCAAGTCGCTCAGGTATGCCCACAGAGCGATTCTCAGCTGAGAGGCCTGATGGATCATTCTCTACT TAGC hTRBV12-3hTRBV12-3 215ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCGAAGCATACAGATGCTGGAGTTATCCAGCAGCCCTCA CAGCTGTGTACTTCACCCCGCCATGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGCCACAACTCGAACCCAGG TCTGTGCCAGCACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGAGACT GTTTAGCTGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATChTRBV12-4 hTRBV12-3 216ATGGGCTCCTGGACCCTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGAGTTATCCAGCAGCCCTCA CAGCTGTGTACTTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTAGAACCCAGG TCTGTGCCAGCACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGAGACT GTTTAGCTGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATChTRBV12-5 hTRBV12-3 217ATGGCCACCAGGCTCCTCTGCTGTGTGGTTCTTTGTCTCCTGGGAGAAGAGCTTATAGATGCTAGAGTCACCCAGCAGCCCTCA CAGCTGTGTATTACACCAAGGCACAAGGTGACAGAGATGGGACAAGAAGTAACAATGAGATGTCAGCCAATTTTAGGCCACAATAGAACCCAGG TTTGTGCTAGTGCTGTTTTCTGGTACAGACAGACCATGATGCAAGGACTGGAGTTGCTGGCTTACTTCCGCAACCGGGCTCCTCTAGGACT GTTTGGTATGATTCGGGGATGCCGAAGGATCGATTCTCAGCAGAGATGCCTGATGCAACTTTAGCCACTCTGAAGATChTRBV13 hTRBV13 218ATGCTTAGTCCTGACCTGCCTGACTCTGCCTGGAACACCAGGCTCCTCTGCCATGTCATGCTTTGTCTCCTGGGAGGAGCTCCTT CAGCCCTGTACTCAGTTTCAGTGGCTGCTGGAGTCATCCAGTCCCCAAGACATCTGATCAAAGAAAAGAGGGAAACAGCCACTCTGGGAGCTGGG TCTGTGCCAGCAAAATGCTATCCTATCCCTAGACACGACACTGTCTACTGGTACCAGCAGGGTCCAGGTCAGGACCCCCAGTTCCTCGGACT GCTTAGGATTTCGTTTTATGAAAAGATGCAGAGCGATAAAGGAAGCATCCCTGATCGATTCTCAGCTCAACAGTTCAGTGACTATCATTCTGAACTGAACAT hTRBV14 hTRBV14 219ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAAGCTGGAGTTACTCAGGGTGCAGCC GATTCTGGAGTTTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAATGCAGAACT TATTTCTGTGCCATCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGAGGAG GCAGCCAAGATGAGTCCGGTATGCCCAACAATCGATTCTTAGCTGAAAGGACTGGAGGGACGTATTCTACTCTGAAhTRBV15 hTRBV15 220ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGGACATCCGC GGGACACAGCCAAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCTCACCAGGC TGTACCTGTGTGATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATCTGG CCACCAGCAGAGGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTT A hTRBV16hTRBV16 221ATGAGCCCAATATTCACCTGCATCACAATCCTTTGTCTGCTGGCTGCAGGTTCTCCTGGTGAAGAAGTCGCCCAGTGAGATCCA GAGGATTCAGCAACTCCAAAACATCTTGTCAGAGGGGAAGGACAGAAAGCAAAATTATATTGTGCCCCAATAAAAGGACACAGTTAGGCTACGAA GTGTATTTTTGTGTGTTTTTTGGTACCAACAGGTCCTGAAAAACGAGTTCAAGTTCTTGATTTCCTTCCAGAATGAAAATGTCTTTGATGCTT CCAGCAGCCAATGAAACAGGTATGCCCAAGGAAAGATTTTCAGCTAAGTGCCTCCCAAATTCACCCTGTAGCCT C hTRBV17hTRBV17 222ATGGATATCTGGCTCCTCTGCTGGGTGACCCTGTGTCTCTTGGCGGCAGGACACTCGGAGCCTGGAGTCAGCCAGGAAGATCCA AGGGACTCAGCCACCCCCAGACACAAGGTCACCAACATGGGACAGGAGGTGATTCTGAGGTGCGATCCATCTTCTGGTCACATGTTTTCCCGCAGA GTGTATCTCTACGTTCACTGGTACCGACAGAATCTGAGGCAAGAAATGAAGTTGCTGATTTCCTTCCAGTACCAAAACATTGCAGTTGCCG AGTAGCGGTGGGATTCAGGGATGCCCAAGGAACGATTCACAGCTGAAAGACCTAACGGAACGTCTTCCACGCT hTRBV18hTRBV18 223ATGGACACCAGAGTACTCTGCTGTGCGGTCATCTGTCTTCTGGGGGCAGGTCTCTCAAATGCCGGCGTCATGCAGGGATCCAGC AGATTCGGCAGCAACCCAAGACACCTGGTCAGGAGGAGGGGACAGGAGGCAAGACTGAGATGCAGCCCAATGAAAGGACACAGTCAGGTAGTGC TTATTTCTGTGCCATGTTTACTGGTATCGGCAGCTCCCAGAGGAAGGTCTGAAATTCATGGTTTATCTCCAGAAAGAAAATATCATAGGAGG AGCTCACCACCATGAGTCAGGAATGCCAAAGGAACGATTTTCTGCTGAATTTCCCAAAGAGGGCCCCAGCATCCTGAhTRBV19 hTRBV19 224ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTTTCCTGGGAGCAAACACCGTGGATGGTGGAATCACTCAGCACTGTGAC AACCCGACAGCTTCCCCAAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCACGATGCATCGGCCCA TTCTATCTCTGTGCATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACTACTCACAGATAGTAAATGACTTTCAAAG CCAGTAGTATAGAGAAAGGAGATATAGCTGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCT A hTRBV20hTRBV20 225ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTACCTGGGAGCTTGGCAGGCTCCGGGCTTGCTGACAGTG CTGAAGACAGCAGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCACCAGTGCC GCTTCTACATCTTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCACATC GCAGTGCTAGAGATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCA TTGTCCACT hTRBV21 hTRBV21 226ATGTGCCTCAGACTTCTCTGCTGTGTGGCCATTTCTTTCTGGGGAGCCAGGCTCCACGGACACCAAGGTCACCCAGAGATCCAG GGGACACAGCACGAGACCTAGACTTCTGGTCAAAGCAAGTGAACAGAAAGCAAAGATGGATTGTGTTCCTATAAAAGCACATAGTTTCCACGGAG TGTATTTCTGTGCATGTTTACTGGTATCGTAAGAAGCTGGAAGAAGAGCTCAAGTTTTTGGTTTACTTTCAGAATGAAGAACTTATTCTCAG CAGCAGCAAAGCAGAAAGCAGAAATAATCAATGAGCGATTTTTAGCCCAATGCTCCAAAAACTCATCCTGTACCTTGhTRBV22 hTRBV22 227ATGGGGAGCTGGGTCCTCTGCTATGTGACCCTGTGTCTCCTGGGAGCAGGACCCTTGGATGCTGACATCTATCAGGTGAAGTTG AACAGCTTTGTAATGCCATTCCAGCTCACTGGGGCTGGATGGGATGTGACTCTGGAGTGGAAACGGAATTTGAGACACAATGACATGCCCACACC CTTCTGTCCTGGGTACTGCTACTGGTACTGGCAGGACCCAAAGCAAAATCTGAGACTGATCTATTACTCAAGGGTTGAAAAGGATAAGCCA GAGCGCACTTCAGAGAGGAGATCTAACTGAAGGCTACGTGTCTGCCAAGAGGAGAAGGGGCTATTTCTTCTCAGGhTRBV23 hTRBV23 228ATGGGCACCAGGCTCCTCGGCTGTGCAGCCCTGTGTCTCCTGGCAGCAGACTCTTTTCATGCCAAAGTCACACAGCCTGGCAAT CCGGGAGACACGACTCCAGGACATTTGGTCAAAGGAAAAGGACAGAAAACAAAGATGGATTGTACCCCCGAAAAAGGACATACTTTCCTGTCCTC GCACTGTATCTCTGTTTATTGGTATCAACAGAATCAGAATAAAGAGTTTATGCTTTTGATTTCCTTTCAGAATGAACAAGTTCTTCAAAGAA TGCGCCAGCAGTGAAACGGAGATGCACAAGAAGCGATTCTCATCTCAATGCCCCAAGAACGCACCCTGCAG CAATChTRBV24 hTRBV24 229ATGGCCTCCCTGCTCTTCTTCTGTGGGGCCTTTTATCTCCTGGGAACAGGGTCCATGGATGCTGATGTTACCCAGAGAGTCTGCC CAGCTCTTTACTTCCCCAAGGAATAGGATCACAAAGACAGGAAAGAGGATTATGCTGGAATGTTCTCAGACTAAGGGTCATGATAGAATCCCCAAC CTGTGCCACCAGATGTACTGGTATCGACAAGACCCAGGACTGGGCCTACGGTTGATCTATTACTCCTTTGATGTCAAAGATATAAACCAGA TGATTTGAAAGGAGAGATCTCTGATGGATACAGTGTCTCTCGACAGGCACAGGCTAAATTCTCCCTGTCCCTAhTRBV25 hTRBV25 230ATGACTATCAGGCTCCTCTGCTACATGGGCTTTTATTTTCTGGGGGCAGGCCTCATGGAAGCTGACATCTACCAGGGAGTCTGC TACCTCTCAGTAACCCCAAGATACCTTGTTATAGGGACAGGAAAGAAGATCACTCTGGAATGTTCTCAAACCATGGGCCATGACAACAGGCCCTC CCTCTGTGCCAGAATGTACTGGTATCAACAAGATCCAGGAATGGAACTACACCTCATCCACTATTCCTATGGAGTTAATTCCACAGAACA CAGTGAATAGAAGGGAGATCTTTCCTCTGAGTCAACAGTCTCCAGAATAAGGACGGAGCATTTTCCCCTGACCCThTRBV26 hTRBV26 231ATGAGCAACAGGCTTCTCTGCTGTGTGATCATTTGTCTCCTAAGAGCAGGCCTCAAGGATGCTGTAGTTACACAAGAAGTCTGC ACATCTGTGTATTTCCCAAGACACAGAATCATTGGGACAGGAAAGGAATTCATTCTACAGTGTTCCCAGAATATGAATCATGTTACACAGCACCAA CTCTATGCCAGCATGTACTGGTATCGACAGGACCCAGGACTTGGACTGAAGCTGGTCTATTATTCACCTGGCACTGGGAGCACTGAACCAG AGTTCATCAAAGGAGATATCTCTGAGGGGTATCATGTTTCTTGAAATACTATAGCATCTTTTCCCCTGACCCThTRBV27 hTRBV27 232ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGGGAGTCGCC ACCTCTCTGTACTAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTACAGCCCCAA TCTGTGCCAGCATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGACCAG GTTTATCTAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCThTRBV28 hTRBV28 233ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGGGAGTCCGC ACATCTATGTACAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAACAGCACCAA CTCTGTGCCAGCTATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGACCAG AGTTTATGAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCThTRBV29 hTRBV29 234ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGGTGAGCAAC CAGCAGCATATAATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCATGAGCCCT TCTCTGCAGCGTGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGGCCACATATGAGAGTGGAGAAGA TGAAGA TTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACThTRBV30 hTRBV30 235ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAGGAGTTCTAA GTGACTCTGGCTCGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATGAAGCTCCT TCTATCTCTGTGCACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGTCTCA CTGGAGTGT AGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCT

What is claimed is:
 1. A method for sequencing immune cell receptorgenes, comprising providing RNA from immune cells; transcribing the RNAinto complementary RNA (cRNA); reverse transcribing the cRNA intocomplementary DNA (cDNA), using one or more primers that comprise afirst adapter sequence, wherein each 5′ end of the cDNA produced byreverse transcription contains the first adapter sequence; amplifyingthe cDNA to produce a first amplification product using a first primerpair comprising a first primer that hybridizes to the first adaptersequence and a second primer that hybridizes to a constant region ofimmune cell receptor gene; amplifying the first amplification product toproduce a second amplification product using a second primer pair, inwhich i. a first primer of the second primer pair binds to the adaptersequence at the 5′ end of the first amplification product, ii. thesecond primer of the second primer pair binds to the constant region ofimmune cell receptor gene in the first amplification product, and iii.the first and second primers comprise adapter sequences for sequencing;and sequencing the second amplification product.
 2. The method accordingto claim 1, wherein the reverse transcription step results in PCRproducts ranging from 150-600 bp.
 3. The method according to claim 1,wherein the immune cell receptor genes are T-cell receptor (TCR) genesor B-cell receptor (BCR) genes.
 4. The method of claim 1, wherein theone or more primers used for reverse transcription hybridize to TCR αchain V segments, optionally wherein the one or more primers used forreverse transcription comprise one or more of SEQ ID NOs: 1-50; orwherein the one or more primers used for reverse transcription hybridizeto TCR β chain V segments, optionally wherein the one or more primersused for reverse transcription comprise one or more of SEQ ID NOs:51-100; or wherein the one or more primers used for reversetranscription hybridize to TCR γ chain V segments; or wherein the one ormore primers used for reverse transcription hybridize to TCR δ chain Vsegments; or wherein the one or more primers used for reversetranscription hybridize to BCR heavy chain V segments; or wherein theone or more primers used for reverse transcription hybridize to BCRlight chain V segments.
 5. The method according to claim 1, wherein theone or more primers used for reverse transcription contain a nucleotidebarcode sequence; optionally wherein the nucleotide barcode comprises 6to 20 nucleotides.
 6. The method according to claim 5, wherein thenucleotide barcode consists of 9 nucleotides; optionally wherein thenucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN orHHHHHNNNN.
 7. The method according to claim 1, wherein the first adaptersequence of the one or more primers used for the reverse transcriptioncomprises a T7 adapter.
 8. The method according to claim 1, wherein theimmune cells are T-cells and wherein the second primer of the first pairof primers hybridizes to the constant region of a TCR gene.
 9. Themethod according to claim 1, wherein the immune cells are B-cells andwherein the second primer of the first pair of primers hybridizes to theconstant region of a BCR gene.
 10. The method according to claim 1,wherein the sequencing is next generation sequencing.
 11. The methodaccording to claim 1, wherein the RNA from the immune cells is obtainedby mixing immune cells with carrier cells before RNA extraction.
 12. Themethod according to claim 1, wherein the immune cells aretumor-infiltrating lymphocytes.
 13. The method according to claim 1,wherein the immune cells are CD4 or CD8 positive T-cells.
 14. The methodaccording to claim 1, wherein the immune cells are purified fromperipheral blood mononuclear cells (PBMC) before RNA extraction.
 15. Themethod according to claim 1, wherein the immune cells are part of amixture of peripheral blood mononuclear cells (PBMC).
 16. The methodaccording to claim 1, wherein the immune cells are derived from amammal; optionally wherein the mammal is a human or a mouse.